From aa52a145fc3dda5d30d9e101747a34cabaf66703 Mon Sep 17 00:00:00 2001 From: SofieVG Date: Thu, 20 Apr 2023 16:31:28 +0200 Subject: [PATCH] Code commit --- 210708_compareSubset.R | 199 ++++++++ 210708_figures.R | 814 +++++++++++++++++++++++++++++++++ 210708_loadDemographics.R | 62 +++ 210708_originalFlowSOMmodels.R | 243 ++++++++++ README.md | 4 + schematic_overview.pdf | Bin 0 -> 525963 bytes 6 files changed, 1322 insertions(+) create mode 100644 210708_compareSubset.R create mode 100644 210708_figures.R create mode 100644 210708_loadDemographics.R create mode 100644 210708_originalFlowSOMmodels.R create mode 100644 schematic_overview.pdf diff --git a/210708_compareSubset.R b/210708_compareSubset.R new file mode 100644 index 0000000..585e2b7 --- /dev/null +++ b/210708_compareSubset.R @@ -0,0 +1,199 @@ +compareSubset <- function(All_traits, subset, names){ + + subset_selected <- ggplot(All_traits %>% dplyr::filter(Group != "QC")) + + geom_point(aes(x = umap_1, y = umap_2, col = names[subset+1][All_traits$Group != "QC"])) + + geom_point(aes(x = umap_1, y = umap_2), col = "black", + data = All_traits %>% dplyr::filter(Group == "QC")) + + theme_void() + + scale_color_discrete(name = "") + + theme(legend.position="bottom") + + diff <- sapply(grep("% *MC", colnames(All_traits), value = TRUE), + function(trait){ + test_res <- wilcox.test(All_traits[subset,trait], + All_traits[!subset,trait]) + fold_change <- median(All_traits[subset,trait])/ + median(All_traits[!subset, trait]) + + return(c(p_value = test_res$p.value, + #`-log10p` = -log10(test_res$p.value), + fold_change = fold_change, + logfoldchange = log10(fold_change))) + }) %>% + t() %>% + data.frame(check.names = FALSE) %>% + tibble::rownames_to_column("Trait") + + diff$p_corrected <- p.adjust(diff$p_value, method = "BH") + diff$`-log10p` <- -log10(diff$p_corrected) + + # max_fold <- max(abs(diff[,"logfoldchange"])) + # max_p <- max(diff[,"-log10p"][!is.infinite(diff[,"-log10p"])], na.rm = TRUE) + # print(max_p) + # subset_volcano <- ggplot(diff) + + # geom_text(aes(x = logfoldchange, + # y = `-log10p`, + # label = Trait), + # size = 2) + + # xlim(-1.5*max_fold,1.5*max_fold)+ + # ylim(0, 1.05*max_p)+ + # theme_minimal() + # + # best_feature <- diff[which.min(diff[,"p_value"]), "Trait"] + # + # subset_bestFeature <- ggplot(All_traits) + + # geom_point(aes_string(x = "subset", y = paste0("`", best_feature, "`*100")), + # position = ggbeeswarm::position_quasirandom(width = 0.3), + # size = 1) + + # geom_boxplot(aes_string(x = "subset", + # y = paste0("`", best_feature, "`*100"), + # col = "subset"), + # outlier.alpha = 0, + # alpha = 0) + + # theme_minimal() + + # ylab(best_feature) + # + # subset_overview <- subset_selected + subset_volcano + subset_bestFeature + # + best_features <- diff[head(order(diff$`-log10p`, + decreasing = TRUE), n = 4), "Trait"] + subset_bestFeature <- list() + + for(best_feature in best_features){ + All_traits[, best_feature] <- All_traits[, best_feature] * 100 + subset_bestFeature[[best_feature]] <- ggplot(All_traits) + + geom_point(aes_string(x = "names[subset+1]", y = paste0("`", best_feature, "`")), + position = ggbeeswarm::position_quasirandom(width = 0.3), + size = 1) + + geom_boxplot(aes_string(x = "names[subset+1]", + y = paste0("`", best_feature, "`"), + col = "names[subset+1]"), + outlier.alpha = 0, + alpha = 0) + + xlab("") + + theme_minimal() + + ylab(best_feature) + + theme(legend.position = "none") + } + + best_correlated <- diff[head(order(diff$`-log10p`, + decreasing = TRUE), n = 10),] + + tnk_sub <- best_correlated[grep("TNK", best_correlated$Trait),] + rownames(tnk_sub) <- gsub("TNK %MC([0-9]*)", "TNK MC\\1", tnk_sub$Trait) + tnk_phenotype <- tnk_mc_mfi[rownames(tnk_sub),] + tnk_heatmap <- pheno_heatmap(tnk_phenotype, tnk_sub[,c("logfoldchange", "-log10p")]) + + bdc_sub <- best_correlated[grep("BDC", best_correlated$Trait),] + rownames(bdc_sub) <- gsub("BDC %MC([0-9]*)", "BDC MC\\1", bdc_sub$Trait) + bdc_phenotype <- bdc_mc_mfi[rownames(bdc_sub),] + bdc_heatmap <- pheno_heatmap(bdc_phenotype, bdc_sub[,c("logfoldchange", "-log10p")]) + + subset_overview <- #subset_volcano + + subset_selected | + ((subset_bestFeature[[1]] + subset_bestFeature[[2]]) / + (subset_bestFeature[[3]] + subset_bestFeature[[4]]) + plot_layout(tag_level = 'new')) | + wrap_plots(grid::grid.grabExpr(ComplexHeatmap::draw(tnk_heatmap)), + #grid::grid.grabExpr(ComplexHeatmap::draw(bdc_heatmap)), # For actual used example no BDC features + ncol = 1) + plot_layout(widths = c(1, 1, 1)) + plot_layout(tag_level = 'new') + return(list(comparison = diff, + plot = subset_overview)) +} + + +compareSubsetAge <- function(All_traits, subset){ + + subset_selected <- ggplot(All_traits) + + geom_point(aes(x = umap_1, y = umap_2, col = subset)) + + theme_void() + + cor <- sapply(grep("% *MC", colnames(All_traits), value = TRUE), + function(trait){ + test_res <- cor.test(All_traits[subset,trait], + All_traits[subset,"Age"], + use = "pairwise.complete.obs", + method = "spearman") + return(c(test_res$estimate["rho"], + p_value = test_res$p.value))#, + # "-log10p" = -log10(test_res$p.value) + }) %>% + t() %>% + data.frame(check.names = FALSE) %>% + tibble::rownames_to_column("Trait") + + cor$p_corrected <- p.adjust(cor$p_value, method = "BH") + cor$`-log10p` <- -log10(cor$p_corrected) + + max_rho <- max(abs(cor[,"rho"])) + max_p <- max(cor[,"-log10p"][!is.infinite(cor[,"-log10p"])], na.rm = TRUE) + print(max_p) + subset_volcano <- ggplot(cor) + + geom_text(aes(x = rho, + y = `-log10p`, + label = Trait), + size = 2) + + xlim(-1.5*max_rho,1.5*max_rho)+ + ylim(0, 1.1* max_p)+ + theme_minimal() + + best_features <- cor[head(order(cor$`-log10p`, + decreasing = TRUE), n = 4), "Trait"] + print(best_features) + subset_bestFeature <- list() + + for(best_feature in best_features){ + All_traits[, best_feature] <- All_traits[, best_feature] * 100 + subset_bestFeature[[best_feature]] <- ggpubr::ggscatter(All_traits[subset,], + x = "Age", + y = best_feature, + add = "reg.line", + add.params = list(color = "#00BFC4"), + conf.int = TRUE, + cor.coef = TRUE, + cor.method = "spearman") + } + + best_correlated <- cor[head(order(cor$`-log10p`, + decreasing = TRUE), n = 10),] + + age_tnk_sub <- best_correlated[grep("TNK", best_correlated$Trait),] + rownames(age_tnk_sub) <- gsub("TNK %MC([0-9]*)", "TNK MC\\1", age_tnk_sub$Trait) + age_tnk_phenotype <- tnk_mc_mfi[rownames(age_tnk_sub),] + age_tnk_heatmap <- pheno_heatmap(age_tnk_phenotype, age_tnk_sub[,c("rho", "-log10p")]) + + age_bdc_sub <- best_correlated[grep("BDC", best_correlated$Trait),] + rownames(age_bdc_sub) <- gsub("BDC %MC([0-9]*)", "BDC MC\\1", age_bdc_sub$Trait) + age_bdc_phenotype <- bdc_mc_mfi[rownames(age_bdc_sub),] + age_bdc_heatmap <- pheno_heatmap(age_bdc_phenotype, age_bdc_sub[,c("rho", "-log10p")]) + + subset_overview <- #subset_volcano + + ((subset_bestFeature[[best_features[1]]] + subset_bestFeature[[best_features[2]]]) / + (subset_bestFeature[[best_features[3]]] + subset_bestFeature[[best_features[4]]]) + plot_layout(tag_level = 'new')) | + wrap_plots(grid::grid.grabExpr(ComplexHeatmap::draw(age_tnk_heatmap)), + grid::grid.grabExpr(ComplexHeatmap::draw(age_bdc_heatmap)), + ncol = 1) + plot_layout(tag_level = 'new') + + return(list(correlation = cor, + plot = subset_overview)) +} + +pheno_heatmap <- function(phenotype, annotation){ + ComplexHeatmap::Heatmap(as.matrix(phenotype), + col = rev(RColorBrewer::brewer.pal(8,"RdBu")), + cluster_columns = FALSE, + cluster_rows = FALSE, + row_names_side = "left", + right_annotation = ComplexHeatmap::rowAnnotation( + df = annotation, + col = list("rho" = circlize::colorRamp2(c(-1, 0, 1), + c("#7b3294", + "white", + "#008837")), + "logfoldchange" = circlize::colorRamp2(c(-1, 0, 1), + c("#7b3294", + "white", + "#008837")), + "-log10p" = circlize::colorRamp2(c(30, 0), + c("#008837", + "white"))))) +} \ No newline at end of file diff --git a/210708_figures.R b/210708_figures.R new file mode 100644 index 0000000..69a35e2 --- /dev/null +++ b/210708_figures.R @@ -0,0 +1,814 @@ +# Load libraries and helper functions ------------------------------------------ + +library(flowCore) +library(FlowSOM) +library(dplyr) +library(ggplot2) +library(patchwork) +source("210708_loadDemographics.R") + +# Set up variables ------------------------------------------------------------- + +recompute <- FALSE +date <- "210708" +#base_dir <- "/auto/net/fs4-Flowcytometry/Storage/u_ysa/Projects/twin_study/52e4c244-0e9b-4573-8e11-d10c249a871c-twinstudy2/" +base_dir <- "D:/Git_repos/twin_study" + +demographics <- loadDemographics(base_dir) +demographics <- dplyr::filter(demographics, `ICS_Viable of CD3` > 25) + +# Load results ----------------------------------------------------------------- + +## TNK + +fsom_TNK <- readRDS(file.path(base_dir, "RDS", paste0(date,"_TNK_FlowSOM.RDS"))) + +result_files_TNK <- list.files(file.path(base_dir, "RDS"), + pattern = paste0(date, "_TNK_Results"), + full.names = TRUE) %>% + stringr::str_sort(numeric = TRUE) + +results_TNK <- lapply(result_files_TNK, readRDS) + +MC_pctgs_TNK <- do.call(rbind, lapply(results_TNK, function(x) x$metacluster_percentages)) +colnames(MC_pctgs_TNK) <- paste0("TNK ", colnames(MC_pctgs_TNK)) + +MC_counts_TNK <- do.call(rbind, lapply(results_TNK, function(x) x$metacluster_counts)) +colnames(MC_counts_TNK) <- paste0("TNK ", colnames(MC_counts_TNK)) + +MC_MFI_TNK <- do.call(rbind, lapply(results_TNK, function(x) x$metacluster_MFIs)) +colnames(MC_MFI_TNK) <- paste0("TNK ", colnames(MC_MFI_TNK)) + +MC_outliers_TNK <- do.call(c, lapply(results_TNK, function(x){ + rowSums(attr(x$cluster_counts, "outliers")) +})) +MC_outliers_TNK <- MC_outliers_TNK / rowSums(MC_counts_TNK) + +## BDC + +fsom_BDC <- readRDS(file.path(base_dir, "RDS", paste0(date,"_BDC_FlowSOM.RDS"))) + +result_files_BDC <- list.files(file.path(base_dir, "RDS"), + pattern = paste0(date, "_BDC_Results_[0-9]"), + full.names = TRUE) %>% + stringr::str_sort(numeric = TRUE) + +results_BDC <- lapply(result_files_BDC, readRDS) + +MC_pctgs_BDC <- do.call(rbind, lapply(results_BDC, function(x) x$metacluster_percentages)) +colnames(MC_pctgs_BDC) <- paste0("BDC ", colnames(MC_pctgs_BDC)) + +MC_counts_BDC <- do.call(rbind, lapply(results_BDC, function(x) x$metacluster_counts)) +colnames(MC_counts_BDC) <- paste0("BDC ", colnames(MC_counts_BDC)) + + +MC_outliers_BDC <- do.call(c, lapply(results_BDC, function(x){ + rowSums(attr(x$cluster_counts, "outliers")) +})) +MC_outliers_BDC <- MC_outliers_BDC / rowSums(MC_counts_BDC) + +## BDC normalised +result_files_BDC_norm <- list.files(file.path(base_dir, "RDS"), + pattern = paste0(date, "_BDC_Results_normalized"), + full.names = TRUE) %>% + stringr::str_sort(numeric = TRUE) + +results_BDC_norm <- lapply(result_files_BDC_norm, readRDS) + +MC_pctgs_BDC_norm <- do.call(rbind, lapply(results_BDC_norm, function(x) x$metacluster_percentages)) +colnames(MC_pctgs_BDC_norm) <- paste0("BDC ", colnames(MC_pctgs_BDC_norm)) + +MC_counts_BDC_norm <- do.call(rbind, lapply(results_BDC_norm, function(x) x$metacluster_counts)) +colnames(MC_counts_BDC_norm) <- paste0("BDC ", colnames(MC_counts_BDC_norm)) + +MC_MFI_BDC_norm <- do.call(rbind, lapply(results_BDC_norm, function(x) x$metacluster_MFIs)) +colnames(MC_MFI_BDC_norm) <- paste0("BDC ", colnames(MC_MFI_BDC_norm)) + + +MC_outliers_BDC_norm <- do.call(c, lapply(results_BDC_norm, function(x){ + rowSums(attr(x$cluster_counts, "outliers")) +})) +MC_outliers_BDC_norm <- MC_outliers_BDC_norm / rowSums(MC_counts_BDC_norm) + +# Combine into one data frame -------------------------------------------------- + +complete <- intersect(rownames(MC_pctgs_BDC_norm), rownames(MC_pctgs_TNK)) +All_traits <- data.frame(demographics[complete,], + MC_pctgs_BDC_norm[complete,], + MC_pctgs_TNK[complete,], + check.names = FALSE) + +counts <- data.frame(demographics[complete,], + MC_counts_BDC_norm[complete,], + MC_counts_TNK[complete,], + check.names = FALSE) + + +date <- "210909" + +# Figures FlowSOM model -------------------------------------------------------- + +for (panel in c("TNK", "BDC")) { + if (panel == "TNK") { + fsom <- fsom_TNK + } else if (panel == "BDC"){ + fsom <- fsom_BDC + } + + # FlowSOMmary + FlowSOMmary(fsom, paste0(date, "_", panel, "_FlowSOM.pdf")) + + # Scatters + channelpairs <- readxl::read_xlsx( + file.path(base_dir, + "Metadata", + paste0("210420_", panel, "_lineage marker combinations for plotting.xlsx")), + col_names = FALSE) + channelpairs <- as.list(as.data.frame(t(channelpairs))) + Plot2DScatters(fsom, + channelpairs = channelpairs, + metaclusters = c(1:40), + plotFile = paste0(date,"_", panel, "_Scatter_MC1-40.png")) +} + +# Figures UMAP ----------------------------------------------------------------- + +# _ UMAP model ----------------------------------------------------------------- + +set.seed(1) +umap <- uwot::umap(scale(All_traits %>% dplyr::select(contains("%"))), + n_neighbors = 10) + +All_traits <- data.frame(All_traits, + umap_1 = umap[,1], + umap_2 = umap[,2], + check.names = FALSE) +All_traits$Cohort[All_traits$Group == "QC"] = "QC" + +ggplot(All_traits) + + geom_point(aes(x = umap_1, y = umap_2, col = Age, shape = Cohort)) + + theme_minimal() + + scale_color_distiller(palette = "RdYlBu") + + +# _ Main overview colored by age ----------------------------------------------- + +umap_age <- ggplot(All_traits %>% dplyr::filter(Group != "QC")) + + geom_point(aes(x = umap_1, y = umap_2, col = Age)) + + geom_point(aes(x = umap_1, y = umap_2), col = "black", + data = All_traits %>% dplyr::filter(Group == "QC")) + + theme_void() + + scale_color_distiller(palette = "RdYlBu") + + theme(legend.position="bottom") + + +# _ Subset highlights ---------------------------------------------------------- + +tnk_mc_mfi <- GetMetaclusterMFIs(fsom_TNK, prettyColnames = TRUE, colsUsed = TRUE) +rownames(tnk_mc_mfi) <- paste0("TNK MC",seq_len(nrow(tnk_mc_mfi))) +tnk_mc_mfi <- tnk_mc_mfi[,stringr::str_sort(colnames(tnk_mc_mfi), numeric = TRUE)] +colnames(tnk_mc_mfi)[2] <- "CD1d" # Too long for plot !! make note in figure legend + +bdc_mc_mfi <- GetMetaclusterMFIs(fsom_BDC, prettyColnames = TRUE, colsUsed = TRUE) +rownames(bdc_mc_mfi) <- paste0("BDC MC",seq_len(nrow(bdc_mc_mfi))) +bdc_mc_mfi <- bdc_mc_mfi[,stringr::str_sort(colnames(bdc_mc_mfi), numeric = TRUE)] + + +source("210708_compareSubset.R") + + +subset_A <- All_traits$umap_1 < -4 +compare_A <- compareSubset(All_traits, subset_A, names = c("Main population", "Subpopulation")) +writexl::write_xlsx(compare_A$comparison, + paste0(date,"_compare_A.xlsx")) +All_traits$subimmunotype <-subset_A + +compare_A$plot +ggsave("compare_subimmunotype.pdf") +writexl::write_xlsx(All_traits, + paste0(date, "_All_traits.xlsx")) + +correlation_all <- compareSubsetAge(All_traits, rep(TRUE, nrow(All_traits))) +writexl::write_xlsx(correlation_all$correlation, + paste0(date,"_correlation_all.xlsx")) + +# Main figure ------------------------------------------------------------------ +schematic <- png::readPNG("schematicOverview_simplified.png", native = TRUE) + +wrap_elements(schematic) / +(umap_age + correlation_all$plot + plot_layout(widths = c(1, 2))) / + (compare_A$plot) + # + plot_layout(tag_level = 'new') + patchwork::plot_annotation(tag_levels = c("A")) +ggsave(paste0(date, "_main_overview.pdf"), + width = 20, height = 15) + +umap_age + +# Figure patient heatmap ------------------------------------------------------- + +dev.off() +pdf(paste0(date, "_patient_heatmap.pdf"), width = 20, height = 60) +pheatmap::pheatmap(All_traits[,grep("%", colnames(All_traits))], + scale = "column", + breaks = seq(-5,5, length.out = 100), + annotation_row = data.frame("QC" = (All_traits[,"Cohort"] == "QC") + 1, + "Subset A" = as.numeric(subset_A), + # "Subset C" = as.numeric(subset_C), + # "Subset B" = as.numeric(subset_B), + (All_traits[,"CMV", drop = FALSE] > 0) + 1, + All_traits[,rev(c("Run#", "Age", "Gender"))], + check.names = FALSE)) +dev.off() + +# Figure supplementary demographics -------------------------------------------- + +umap_gender <- ggplot(All_traits) + + geom_point(aes(x = umap_1, y = umap_2, col = Gender)) + + theme_void() + +umap_CMV <- ggplot(All_traits[c(which(is.na(All_traits$CMV)),which(!is.na(All_traits$CMV))),]) + + geom_point(aes(x = umap_1, y = umap_2, col = CMV > 0)) + + theme_void() + + scale_color_manual(values = c("TRUE" = "#d95f02", "FALSE" = "#7570b3"), + na.value = "lightgrey") + +umap_Run <- ggplot(All_traits) + + geom_point(aes(x = umap_1, y = umap_2, col = `Run#`)) + + geom_text(aes(x = umap_1, y = umap_2, label = `Run#`), col = "red", + data = dplyr::filter(All_traits, Cohort == "QC")) + + theme_void() + +(umap_age + umap_gender) / (umap_CMV + umap_Run) + + plot_annotation(title = "Demographic information", + subtitle = paste0("Including ", nrow(All_traits), " samples,", + " visualised with UMAP using 80 phenotypic features.")) + +ggsave(paste0(date,"_demographics.pdf"), + width = 20, height = 15) + +# Figures stability ------------------------------------------------------------ + +distances_longitudinal_HD <- All_traits[All_traits$Group == "Longitudinal",] %>% + group_by(`Sample ID`) %>% + summarise(Distance = mean(dist(cbind(across(contains("%"))))), + Time = max(`Weeks from first timepoint`)) + +distances_longitudinal_ref <- All_traits[All_traits$Group == "Longitudinal",] %>% + mutate(`Sample ID` = sample(`Sample ID`)) %>% + group_by(`Sample ID`) %>% + summarise(Distance = mean(dist(cbind(across(contains("%"))))), + Time = max(`Weeks from first timepoint`)) + +distances_twins <- All_traits[All_traits$Cohort == "UK",] %>% + dplyr::group_by(`New Family ID`, `Zygosity`) %>% + dplyr::summarise(Distance = mean(dist(cbind(across(contains("%")))))) + +distances_twins_ref <- All_traits[All_traits$Cohort == "UK",] %>% + mutate(`New Family ID` = sample(`New Family ID`)) %>% + dplyr::group_by(`New Family ID`) %>% + dplyr::summarise(Distance = mean(dist(cbind(across(contains("%")))))) + +pdf(paste0(date, "_Distances_longitudinal_HD.pdf")) +plot(density(na.omit(distances_longitudinal_ref$Distance)), + ylim = c(0,8), + main = "Distances HD", + xlab = "Distance") +lines(density(na.omit(distances_longitudinal_HD$Distance)), col = "red") +lines(density(na.omit(distances_twins$Distance)), col = "cyan") +lines(density(na.omit(distances_twins %>% + dplyr::filter(Zygosity == "MZ") %>% + dplyr::pull(Distance))), col = "blue") +lines(density(na.omit(distances_twins %>% + dplyr::filter(Zygosity == "DZ") %>% + dplyr::pull(Distance))), col = "green") +lines(density(na.omit(distances_twins_ref$Distance)), col = "grey") +legend("topright", + legend = c("longitudinal","longitudinal_scrambled", + "twin", "twin_MZ", "twin_DZ", "twin_scrambled"), + col = c("red", "black", + "cyan", "blue", "green", "grey"), + lty = 1) +dev.off() + +distances_longitudinal_2D <- All_traits[All_traits$Group == "Longitudinal",] %>% + group_by(`Sample ID`) %>% + summarise(Distance = mean(dist(cbind(across(contains("umap"))))), + Time = max(`Weeks from first timepoint`)) + +distances_longitudinal <- merge(distances_longitudinal_HD, + distances_longitudinal_2D, + by = "Sample ID", + suffixes = c("_HD", "_2D")) + +cor_dist_long <- cor(distances_longitudinal$Distance_HD, + distances_longitudinal$Distance_2D, + use = "complete.obs") + +ggplot(distances_longitudinal, + aes(x = Distance_HD, y = Distance_2D)) + + geom_point() + theme_minimal() + + ggtitle(paste0("Correlation HD vs 2D: ", round(cor_dist_long, 2))) +ggsave(paste0(date,"_Distances_longitudinal_HDvs2D.pdf")) + + + +distances_longitudinal <- All_traits[All_traits$Group == "Longitudinal",] %>% + group_by(`Sample ID`) %>% + summarise(Distance = mean(dist(cbind(.data[["umap_1"]], + .data[["umap_2"]]))), + Time = max(.data[["Weeks from first timepoint"]])) + +threshold <- 1 + +density_longitudinal <- ggplot(distances_longitudinal) + + geom_histogram(aes(Distance), binwidth = 0.2) + + geom_vline(aes(xintercept = threshold)) + + theme_minimal() +distance_count <- table(distances_longitudinal$Distance <= threshold) + +to_plot <- merge(All_traits, distances_longitudinal, + by = "Sample ID", all.x = TRUE) +writexl::write_xlsx(to_plot, + "distances_longitudinal.xlsx") + +umap_longitudinal <- + ggplot(to_plot[c(which(is.na(to_plot$Distance)),which(!is.na(to_plot$Distance))),] ) + + geom_point(aes(x = umap_1, y = umap_2, col = Distance)) + # as.factor(`Run#`))) + + geom_line(aes(x = umap_1, y = umap_2, group = as.factor(`Sample ID`), col = Distance), + data = to_plot[!is.na(to_plot$Distance), ]) + + scale_color_distiller(palette = "RdYlBu", na.value = "lightgrey") + + theme_minimal() + + ggtitle(paste0(distance_count["FALSE"], " distances larger than ", threshold)) + +umap_longitudinal_smallDistanceOnly <- + ggplot(to_plot[c(which(is.na(to_plot$Distance)),which(!is.na(to_plot$Distance))),] ) + + geom_point(aes(x = umap_1, y = umap_2, col = Distance)) + + #geom_point(aes(x = umap_1, y = umap_2, col = as.factor(`Run#`))) + + geom_line(aes(x = umap_1, y = umap_2, group = as.factor(`Sample ID`), col = Distance), + data = to_plot[!is.na(to_plot$Distance) & to_plot$Distance <= threshold, ]) + + scale_color_distiller(palette = "RdYlBu", na.value = "lightgrey") + + theme_minimal() + + ggtitle(paste0(distance_count["TRUE"], " distances smaller or equal to ", threshold)) + + +density_longitudinal / (umap_longitudinal + umap_longitudinal_smallDistanceOnly ) + +ggsave(paste0(date, "_longitudinalChanges.pdf"), + width = 20, height = 11.6) + + + +distances_twins <- All_traits[All_traits$Cohort == "UK",] %>% + dplyr::group_by(`New Family ID`, `Zygosity`) %>% + dplyr::summarise(Distance = mean(dist(cbind(.data[["umap_1"]], .data[["umap_2"]])))) + +density_twins <- ggplot(distances_twins) + + geom_histogram(aes(Distance, fill = Zygosity), binwidth = 0.1) + + geom_vline(aes(xintercept = threshold)) + + theme_minimal() +distance_twins_count <- table(distances_twins$Distance <= threshold) + +umap_twins <- ggplot(All_traits ) + + geom_point(aes(x = umap_1, y = umap_2, col = as.factor(`Zygosity`))) + + geom_line(aes(x = umap_1, y = umap_2, col = as.factor(`Zygosity`), + group = as.factor(`New Family ID`)), + data = All_traits[All_traits$Cohort == "UK",]) + + theme_minimal() + + +density_twins / umap_twins + +ggsave(paste0(date, "_twinChanges.pdf"), + width = 20, height = 11.6) + + + + +# Figures CV ------------------------------------------------------------------- + +# _ Frequency ---- + +All_traits_stacked <- tidyr::pivot_longer(All_traits, + cols = grep("%", colnames(All_traits), value = TRUE), + names_to = "Trait", + values_to = "Frequency") +All_traits_stacked$Panel <- stringr::str_sub(All_traits_stacked$Trait, 1, 3) + +Frequency_plot <- ggplot(All_traits_stacked, + aes(x = reorder(Trait, Frequency, FUN = mean), + y = log10(Frequency*100))) + + geom_jitter(width = 0.2, size = 0.1, alpha = 0.3, color = "gray60") + + geom_boxplot(fatten = NULL, outlier.shape = NA, coef = 0, fill = "white") + # fatten = NULL to make midline disappear, causes warnings which can be safely ignored + # geom_boxplot(All_traits_stacked %>% dplyr::filter(Cohort == "QC"), + # mapping = aes(x = Trait, + # y = log10(Frequency*100), + # fill = Panel, color = Panel), + # width = 0.4, + # fatten = 0, outlier.shape = NA, coef = 0) + + geom_point(All_traits_stacked %>% dplyr::filter(Cohort == "QC"), + mapping = aes(x = Trait, + y = log10(Frequency*100), + fill = Panel, color = Panel), + position = ggbeeswarm::position_quasirandom()) + + annotation_logticks(sides = c("l"), scaled = TRUE) + + theme_classic() + + xlab("Immune trait") + ylab("Frequency (log10 %)") + + theme(axis.text.x = element_text(size = 7, angle = 90, vjust = 0.5, hjust=1), + legend.position = "none") + + scale_color_manual(values = c("BDC" = "#F8766D", + "ICS" = "#00BA38", + "TNK" = "#619CFF")) + + scale_fill_manual(values = c("BDC" = "#F8766D", + "ICS" = "#00BA38", + "TNK" = "#619CFF")) +Frequency_plot + +# _ QC CV vs count ---- + +CV_QC <- All_traits_stacked %>% + dplyr::filter(Cohort == "QC") %>% + group_by(Trait) %>% + dplyr::summarize(Panel = unique(Panel), + SD = sd(Frequency, na.rm=TRUE), + Mean = mean(Frequency, na.rm=TRUE)) %>% + mutate(CV = SD/Mean * 100) + +CV_QC$Count <- sapply(CV_QC$Trait, + function(trait){ + mean(counts[counts$Group == "QC", + gsub("%", "", trait)])}) + +Poison_fun <- function(x) ((x^(1/2))/x)*100 + +CV_freq_vs_count <- ggplot(CV_QC[sample(nrow(CV_QC)), ], + aes(y = `CV`, x = `Count`, color = `Panel`)) + + stat_function(fun = Poison_fun, geom = "area", color = "gray", fill = "gray") + + geom_point(size = 2, alpha = 0.5) + + scale_x_log10(breaks = scales::trans_breaks("log10", function(x) 10^x), + labels = scales::trans_format("log10", scales::math_format(10^.x)), + expand = c(0, 0), + #limits = c(0.05, 200000)) + + limits = c(1, 200000)) + + #scale_y_continuous(limits = c(0,450), expand = c(0, 0)) + + scale_y_continuous(limits = c(0,150), expand = c(0, 0)) + + annotation_logticks(sides = c("b"), scaled = TRUE) + + coord_cartesian(clip = 'off') + + geom_hline(yintercept = 30, color = "darkred", linetype="dashed") + + ylab("%CV Frequency QC samples") + + xlab("Mean cell count QC samples (log)") + + theme_classic() + + theme(legend.position = "none")+ + scale_color_manual(values = c("BDC" = "#F8766D", + "ICS" = "#00BA38", + "TNK" = "#619CFF")) + + scale_fill_manual(values = c("BDC" = "#F8766D", + "ICS" = "#00BA38", + "TNK" = "#619CFF")) + +CV_freq_vs_count + +# _ CV all ---- + +All_traits_nomultiples_stacked <- All_traits_stacked %>% #Extract only weeks 0 and NA + dplyr::filter(`Weeks from first timepoint` == 0 | + is.na(`Weeks from first timepoint`) & + duplicated == 0) + +#Calculate Mean, SD and %CV for Frequency traits of all sample +CV_nomultiples_total <- All_traits_nomultiples_stacked %>% + group_by(Trait) %>% + dplyr::summarize(Panel = unique(Panel), + SD_total = sd(Frequency, na.rm=TRUE), + Mean_total = mean(Frequency, na.rm=TRUE)) %>% + mutate(CV_total = SD_total/Mean_total * 100) + +#Calculate CV values for just twinsUK +CV_nomultiples_TwinsUK <- All_traits_nomultiples_stacked %>% + dplyr::filter(Cohort == "UK") %>% + group_by(Trait) %>% + dplyr::summarize(Panel = unique(Panel), + SD_Twins = sd(Frequency, na.rm=TRUE), + Mean_Twins = mean(Frequency, na.rm=TRUE)) %>% + mutate(CV_Twins = SD_Twins/Mean_Twins * 100) + +#Calculate CV values for just VRC +CV_nomultiples_VRC <- All_traits_nomultiples_stacked %>% + dplyr::filter(Cohort == "VRC") %>% + group_by(Trait) %>% + dplyr::summarize(Panel = unique(Panel), + SD_VRC = sd(Frequency, na.rm=TRUE), + Mean_VRC = mean(Frequency, na.rm=TRUE)) %>% + mutate(CV_VRC = SD_VRC/Mean_VRC * 100) + + +colnames(CV_QC)[3:7] <- paste0(colnames(CV_QC)[3:7], "_QC") + +CV_nomultiples <- CV_nomultiples_total %>% + inner_join(CV_nomultiples_TwinsUK, by = c("Trait", "Panel")) %>% + inner_join(CV_nomultiples_VRC, by = c("Trait", "Panel")) %>% + inner_join(CV_QC, by = c("Trait", "Panel")) + + +PlotCVComparison <- function(CV_nomultiples, + var1, + var2, + lab1, + lab2, + title){ + ggplot(CV_nomultiples, + aes_string(x = var1, y = var2)) + + geom_rect(aes(xmin=0, xmax=30, ymin=0, ymax=Inf), fill = "gray80") + + geom_rect(aes(xmin=0, xmax=Inf, ymin=0, ymax=30), fill = "gray80") + + geom_point(aes(color = Panel)) + + scale_x_continuous(limits = c(0, 200), expand = c(0, 0)) + + scale_y_continuous(limits = c(0,200), expand = c(0, 0)) + + scale_color_manual(values = c("BDC" = "#F8766D", + "ICS" = "#00BA38", + "TNK" = "#619CFF")) + + xlab(lab1) + + ylab(lab2) + + ggtitle(title) + + geom_abline(intercept = 0, slope = 1, color = "darkred", linetype = "dashed") + + theme_classic() +} + +#Plot CV total vs QC +CV1 <- PlotCVComparison(CV_nomultiples, + "CV_QC", "CV_total", + "%CV - QC Sample", "%CV - All samples", + "QV vs all samples") + +CV2 <- PlotCVComparison(CV_nomultiples, + "CV_Twins", "CV_VRC", + "%CV - Twins UK cohort", "%CV - VRC cohort", + "TwinsUK vs VRC") + +# _ Overview figure ---- + +Frequency_plot / (CV_freq_vs_count + CV1 + CV2) +ggsave(paste0(date, "_CV_overview.pdf"), + width = 20, height = 11.6) + + +# Figures batch effect --------------------------------------------------------- + +set.seed(1) +umap_TNK <- uwot::umap(scale(All_traits %>% dplyr::select(contains("TNK %"))), + n_neighbors = 10) +umap_BDC_orig <- uwot::umap(scale(MC_pctgs_BDC[complete, ]), + n_neighbors = 10) +umap_BDC_norm <- uwot::umap(scale(All_traits %>% dplyr::select(contains("BDC %"))), + n_neighbors = 10) + + +All_traits[, c("umap_tnk_1", "umap_tnk_2", + "umap_bdc_o_1", "umap_bdc_o_2", + "umap_bdc_n_1", "umap_bdc_n_2")] <- cbind(umap_TNK, + umap_BDC_orig, + umap_BDC_norm) + +set.seed(1) +p_umap_tnk <- ggplot(All_traits[sample(nrow(All_traits)),]) + + geom_point(aes(x = umap_tnk_1, y = umap_tnk_2, col = `Run#`)) + + theme_void() + + scale_color_distiller(palette = "RdYlBu") + + ggtitle("UMAP based on 40 TNK traits") + +p_umap_bdc <- ggplot(All_traits[sample(nrow(All_traits)),]) + + geom_point(aes(x = umap_bdc_o_1, y = umap_bdc_o_2, col = `Run#`)) + + theme_void() + + scale_color_distiller(palette = "RdYlBu") + + ggtitle("UMAP based on 40 BDC traits - original") + +p_umap_bdc_n <- ggplot(All_traits[sample(nrow(All_traits)),]) + + geom_point(aes(x = umap_bdc_n_1, y = umap_bdc_n_2, col = `Run#`)) + + theme_void() + + scale_color_distiller(palette = "RdYlBu") + + ggtitle("UMAP based on 40 BDC traits - normalized") + +(p_umap_tnk + p_umap_bdc + p_umap_bdc_n) +ggsave(paste0(date, "_umap_separate.pdf"), + width = 20, height = 5) + +# demographics %>% +# filter(Group == "QC") %>% +# arrange("Run #") %>% +# pull(`FlowJo ID`) %>% +# paste0("preprocessed_BDC/", . ,".fcs") %>% +# FlowSOM::PlotFileScatters(channels = GetChannels(fsom_BDC, +# c("CD123", "CD11c")), +# names = paste0("Run ", 1:19), +# plotFile = "QC_CD123_CD11c.png", +# +# ncol = 2, +# nrow = 1) + +norm_model <- readRDS("RDS/CytoNorm_model_BDC.RDS") + +#plot(norm_model$clusterRes$`1`$splines$`1`$`B610-A` + +plotSpline <- function(norm_model, cluster, batch, channel, + min = 0, max = 8){ + graphics::plot(norm_model$clusterRes[[cluster]]$quantiles[[batch]][,channel], + norm_model$clusterRes[[cluster]]$refQuantiles[,channel], + xlim = c(min, max), ylim = c(min, max), pch = 19, + bty = "n", xaxt = "n", yaxt = "n", + xlab = "", ylab = "", main = FlowSOM::GetMarkers(norm_model$fsom, channel)) + graphics::lines(c(min, max), c(min, max), col = "#999999") + x <- seq(min, max, 0.1) + graphics::lines(x, norm_model$clusterRes[[cluster]]$splines[[batch]][[channel]](x), + col = "#b30000") +} + + +png(paste0(date, "_splines_overview.png"), + width = 10 * 200, height = 17 * 200) +graphics::layout(matrix(seq_len(10*17), ncol = 10)) +graphics::par(mar = c(1, 1, 5, 0)) +for(cluster in names(norm_model$clusterRes)){ + message(Sys.time(),": Cluster ",cluster) + for(channel in names(norm_model$fsom$prettyColnames[1:17])){ + plotSpline(norm_model, + cluster = cluster, + batch = 2, + channel = channel, + min = -1, max = 5) + } +} +dev.off() + +# MFI -------------------------------------------------------------------------- + +MFI_traits <- data.frame(demographics[complete,], + MC_MFI_BDC_norm[complete,], + MC_MFI_TNK[complete,], + check.names = FALSE) + +MFI_traits_stacked <- tidyr::pivot_longer(MFI_traits, + cols = grep("MC", colnames(MFI_traits), value = TRUE), + names_to = "Trait", + values_to = "MFI") +MFI_traits_stacked$Panel <- stringr::str_sub(MFI_traits_stacked$Trait, 1, 3) + +MFI_plot <- + MFI_traits_stacked %>% + mutate(Trait = forcats::fct_reorder(Trait, MFI, median, na.rm = TRUE)) %>% + ggplot(aes(x = Trait, y = MFI)) + + #geom_jitter(width = 0.2, size = 0.1, alpha = 0.3, color = "gray60") + + geom_boxplot(fatten = NULL, outlier.shape = NA, coef = 0, fill = "white") + # fatten = NULL to make midline disappear, causes warnings which can be safely ignored + geom_point(MFI_traits_stacked %>% dplyr::filter(Group == "QC"), + mapping = aes(x = Trait, + y = MFI, + fill = Panel, color = Panel), + position = ggbeeswarm::position_quasirandom(), + size = 0.5) + + theme_classic() + + xlab("Immune trait") + ylab("MFI (biexponentially transformed)") + + theme(axis.text.x = element_text(size = 7, angle = 90, vjust = 0.5, hjust=1), + legend.position = "none") + + scale_color_manual(values = c("BDC" = "#F8766D", + "ICS" = "#00BA38", + "TNK" = "#619CFF")) + + scale_fill_manual(values = c("BDC" = "#F8766D", + "ICS" = "#00BA38", + "TNK" = "#619CFF")) +MFI_plot +ggsave(paste0(date, "_MFI_traits.pdf"), + width = 65, height = 8, limitsize = FALSE) + +# _ CV ---- + +CV_MFI_QC <- MFI_traits_stacked %>% + dplyr::filter(Group == "QC") %>% + group_by(Trait) %>% + dplyr::summarize(Panel = unique(Panel), + SD = sd(MFI, na.rm=TRUE), + Mean = mean(MFI, na.rm=TRUE)) %>% + mutate(CV = SD/Mean * 100) + +CV_MFI_QC$Count <- sapply(CV_MFI_QC$Trait, + function(trait){ + mean(counts[counts$Group == "QC", + gsub("(.*) (.*) (.*)", "\\1 \\2", trait)])}) +CV_MFI_freq_vs_count <- ggplot(CV_MFI_QC[sample(nrow(CV_MFI_QC)), ], + aes(y = `CV`, x = `Count`, color = `Panel`)) + + stat_function(fun = Poison_fun, geom = "area", color = "gray", fill = "gray") + + geom_point(size = 2, alpha = 0.5) + + scale_x_log10(breaks = scales::trans_breaks("log10", function(x) 10^x), + labels = scales::trans_format("log10", scales::math_format(10^.x)), + expand = c(0, 0), + #limits = c(0.05, 200000)) + + limits = c(1, 200000)) + + #scale_y_continuous(limits = c(0,450), expand = c(0, 0)) + + scale_y_continuous(limits = c(0,150), expand = c(0, 0)) + + #annotation_logticks(sides = c("b"), scaled = TRUE) + + coord_cartesian(clip = 'off') + + geom_hline(yintercept = 30, color = "darkred", linetype="dashed") + + ylab("%CV MFI QC samples") + + xlab("Mean cell count QC samples (log)") + + theme_classic() + + theme(legend.position = "none")+ + scale_color_manual(values = c("BDC" = "#F8766D", + "ICS" = "#00BA38", + "TNK" = "#619CFF")) + + scale_fill_manual(values = c("BDC" = "#F8766D", + "ICS" = "#00BA38", + "TNK" = "#619CFF")) + +CV_MFI_freq_vs_count + + +MFI_traits_nomultiples_stacked <- MFI_traits_stacked %>% #Extract only weeks 0 and NA + dplyr::filter(`Weeks from first timepoint` == 0 | + is.na(`Weeks from first timepoint`) & + duplicated == 0) + +#Calculate Mean, SD and %CV for MFI traits of all sample +CV_MFI_nomultiples_total <- MFI_traits_nomultiples_stacked %>% + group_by(Trait) %>% + dplyr::summarize(Panel = unique(Panel), + SD_total = sd(MFI, na.rm=TRUE), + Mean_total = mean(MFI, na.rm=TRUE)) %>% + mutate(CV_total = SD_total/Mean_total * 100) + +#Calculate CV values for just twinsUK +CV_MFI_nomultiples_TwinsUK <- MFI_traits_nomultiples_stacked %>% + dplyr::filter(Cohort == "UK") %>% + group_by(Trait) %>% + dplyr::summarize(Panel = unique(Panel), + SD_Twins = sd(MFI, na.rm=TRUE), + Mean_Twins = mean(MFI, na.rm=TRUE)) %>% + mutate(CV_Twins = SD_Twins/Mean_Twins * 100) + +#Calculate CV values for just VRC +CV_MFI_nomultiples_VRC <- MFI_traits_nomultiples_stacked %>% + dplyr::filter(Cohort == "VRC") %>% + group_by(Trait) %>% + dplyr::summarize(Panel = unique(Panel), + SD_VRC = sd(MFI, na.rm=TRUE), + Mean_VRC = mean(MFI, na.rm=TRUE)) %>% + mutate(CV_VRC = SD_VRC/Mean_VRC * 100) + + +colnames(CV_MFI_QC)[3:7] <- paste0(colnames(CV_MFI_QC)[3:7], "_QC") + +CV_MFI_nomultiples <- CV_MFI_nomultiples_total %>% + inner_join(CV_MFI_nomultiples_TwinsUK, by = c("Trait", "Panel")) %>% + inner_join(CV_MFI_nomultiples_VRC, by = c("Trait", "Panel")) %>% + inner_join(CV_MFI_QC, by = c("Trait", "Panel")) + +CV_MFI_1 <- PlotCVComparison(CV_MFI_nomultiples, + "CV_QC", "CV_total", + "%CV - QC Sample", "%CV - All samples", + "QV vs all samples") + +CV_MFI_2 <- PlotCVComparison(CV_MFI_nomultiples, + "CV_Twins", "CV_VRC", + "%CV - Twins UK cohort", "%CV - VRC cohort", + "TwinsUK vs VRC") + +CV_MFI_freq_vs_count + CV_MFI_1 + CV_MFI_2 +ggsave(paste0(date, "_MFI_CV.pdf"), + width = 20, height = 6) + +# Outliers ---- + +outliers <- data.frame(demographics[complete,], + outliers_TNK = MC_outliers_TNK[complete], + outliers_BDC_original = MC_outliers_BDC[complete], + outliers_BDC = MC_outliers_BDC_norm[complete], + check.names = FALSE) +p_outliers_TNK <- ggplot(outliers) + + geom_point(aes(x = reorder(`FlowJo ID`, `Run#`), + y = outliers_TNK * 100, + col = `Run#`)) + + scale_color_distiller(palette = "RdYlBu") + + ylim(0,30) + +p_outliers_BDC_o <- ggplot(outliers) + + geom_point(aes(x = reorder(`FlowJo ID`, `Run#`), + y = outliers_BDC_original * 100, + col = `Run#`)) + + scale_color_distiller(palette = "RdYlBu") + + ylim(0,30) + +p_outliers_BDC <- ggplot(outliers) + + geom_point(aes(x = reorder(`FlowJo ID`, `Run#`), + y = outliers_BDC * 100, + col = `Run#`)) + + scale_color_distiller(palette = "RdYlBu") + + ylim(0,30) + +p_outliers_norm <- ggplot(outliers) + + geom_point(aes(x = outliers_BDC_original * 100, + y = outliers_BDC * 100, + col = `Run#`)) + + scale_color_distiller(palette = "RdYlBu") + + theme_minimal() + + geom_abline(aes(slope = 1, intercept = 0)) + +(p_outliers_TNK / p_outliers_BDC_o / p_outliers_BDC) | p_outliers_norm + +ggsave(paste0(date, "_outliers.pdf"), + width = 20, height = 11.6) + diff --git a/210708_loadDemographics.R b/210708_loadDemographics.R new file mode 100644 index 0000000..68019b8 --- /dev/null +++ b/210708_loadDemographics.R @@ -0,0 +1,62 @@ +library(dplyr) + +loadDemographics <- function(base_dir){ + demographics <- read.csv(paste0(base_dir, "/Metadata/200901_Twin_demographics_adjusted.csv"), + check.names = FALSE, + stringsAsFactors = FALSE) + demographics$`FlowJo ID` <- as.character(demographics$`FlowJo ID`) + demographics$`Sample ID` <- NULL # Remove, does not contain full information + + # Add run + run <- read.csv(paste0(base_dir, "/Metadata/201122_FlowJo ID and Run.csv"), + check.names = FALSE) + rownames(run) <- run$`FlowJo ID` <- as.character(run$`FlowJo ID`) + demographics <- merge(demographics, run, + by = "FlowJo ID", + all = TRUE, sort = FALSE) + + # Add sample ID + sampleID <- read.csv(paste0(base_dir, "/Metadata/201122_FlowJo and Sample ID.csv"), + check.names = FALSE) + rownames(sampleID) <- sampleID$`FlowJo ID` <- as.character(sampleID$`FlowJo ID`) + demographics <- merge(demographics, sampleID, + by = c("FlowJo ID"), + all = TRUE, sort = FALSE) + + # Add CMV status + CMV_VRC <- read.csv(file.path(base_dir, "Metadata/2021-06-24 VRC serostatus for EBV CMV and HSV1_2.csv"), + check.names = FALSE, + stringsAsFactors = FALSE) + CMV_VRC <- CMV_VRC %>% dplyr::filter(`Sample ID` != "S3-0860-01") + CMV_twins <- read.csv(file.path(base_dir, "Metadata/2021-06-24 Twins serostatus for EBV CMV and HSV1_2.csv"), + check.names = FALSE, + stringsAsFactors = FALSE) + CMV <- rbind(CMV_VRC, CMV_twins) + CMV[is.na(CMV)] <- 0 + demographics <- merge(demographics, CMV, + by = c("Sample ID"), + all.x = TRUE, sort = FALSE) + + # Add viability + viability <- sapply(c("BDC", "TNK", "ICS"), + function(sheet) readxl::read_xlsx(file.path(base_dir, + "Metadata/210808_Viability summary_updated.xlsx"), + sheet = sheet) %>% as.data.frame()) + colnames(viability$ICS)[3:5] <- paste0("ICS_", colnames(viability$ICS)[3:5]) + viability_all <- merge(viability$BDC[,2:3], + viability$TNK[,2:3], + by = "FlowJo ID", + suffixes = c("_BDC", "_TNK"), + all = TRUE) + viability_all <- merge(viability_all, + viability$ICS[2:5], + by = "FlowJo ID", + all = TRUE) + colnames(viability_all) + demographics <- merge(demographics, viability_all, + by = c("FlowJo ID"), + all.x = TRUE, sort = FALSE) + + rownames(demographics) <- demographics$`FlowJo ID` + demographics +} diff --git a/210708_originalFlowSOMmodels.R b/210708_originalFlowSOMmodels.R new file mode 100644 index 0000000..0c65e28 --- /dev/null +++ b/210708_originalFlowSOMmodels.R @@ -0,0 +1,243 @@ +devtools::install_github("saeyslab/FlowSOM") +devtools::install_github("saeyslab/CytoNorm") + +# Load libraries and helper functions ------------------------------------------ + +library(flowCore) +library(FlowSOM) +source("twin_study/210708_loadDemographics.R") + +# Set up variables ------------------------------------------------------------- + +recompute <- FALSE +date <- "210708" +base_dir <- "/auto/net/fs4-Flowcytometry/Storage/u_ysa/Projects/twin_study/52e4c244-0e9b-4573-8e11-d10c249a871c-twinstudy2/" + +demographics <- loadDemographics(base_dir) +demographics <- dplyr::filter(demographics, `ICS_Viable of CD3` > 25) +training_set <- demographics[demographics[,"Run#"] %in% c(1,2), "FlowJo ID"] + +# Panel specific variables ----------------------------------------------------- + +panel <- "BDC" +#panel <- "TNK" + +# Raw files +fcs_path <- file.path(base_dir, panel, "fcs files_comped and pregated") + +# New directories and files which will be created by the script +preprocessed_path <- file.path(base_dir, paste0(date, "_", panel, "_preprocessed")) +agg_path <- file.path(base_dir, "RDS", paste0(date, "_", panel, "_aggregate.fcs")) +fsom_path <- file.path(base_dir, "RDS", paste0(date, "_", panel, "_FlowSOM.RDS")) +output_path <- file.path(base_dir, "RDS", paste0(date, "_", panel, "_Results.RDS")) + +# Markers to use +marker_files <- c("TNK" = "210203_TNK_Marker and Population for FlowSOM.xlsx", + "BDC" = "210420_BDC_Marker and subset list.xlsx") + +marker_overview <- readxl::read_xlsx(file.path(base_dir, "Metadata", + marker_files[panel]), + sheet = 1) + +channels_to_use <- marker_overview$Channel[marker_overview[[4]] %in% c("Lineage", "Subset identification")] +channels_to_use <- paste0(channels_to_use, "-A") +channels_to_use <- gsub("U395-A", "U390-A", channels_to_use) +channels_to_use <- gsub("U670-A", "U660-A", channels_to_use) + +channels_MFI <- marker_overview$Channel[marker_overview[[4]] %in% c("Functional")] +channels_MFI <- paste0(channels_MFI, "-A") +channels_MFI <- gsub("U395-A", "U390-A", channels_MFI) +channels_MFI <- gsub("U670-A", "U660-A", channels_MFI) + +# Preprocessing preparation ---------------------------------------------------- + +# Manually defined BDC gate +BDC_gate <- flowCore::polygonGate(filterId = "CD14 or HLADR positive", + .gate = matrix(c(-1, 2, + 1.8, 2, + 1.8, -1, + 4, -1, + 4, 4, + -1, 4), + ncol = 2, byrow = TRUE, + dimnames = list(NULL, c("U660-A", "V510-A")))) + +# Load in first file to estimate transformation + +ff <- read.FCS(file.path(fcs_path, "1.fcs")) +ff <- compensate(ff, keyword(ff)[["$SPILLOVER"]]) +tf <- estimateLogicle(ff, + colnames(ff)[4:31]) + +# Preprocess the data ---------------------------------------------------------- +if(!dir.exists(preprocessed_path)) dir.create(preprocessed_path) + +set.seed(1) +for(file in paste0(demographics$`FlowJo ID`, ".fcs")){ + if(file.exists(file.path(fcs_path, file)) & + !file.exists(file.path(preprocessed_path, file))){ + message(Sys.time(),": Preprocessing ", file) + ff <- read.FCS(file.path(fcs_path, file)) + ff <- compensate(ff, keyword(ff)[["$SPILLOVER"]]) + ff <- transform(ff, tf) + if(panel == "BDC") ff <- ff[flowCore::filter(ff, BDC_gate)@subSet,] + write.FCS(ff, + file.path(preprocessed_path, file)) + } +} + +# Make an aggregate of all training samples ------------------------------------ + +if(!recompute & file.exists(agg_path)){ + agg <- read.FCS(agg_path) + message(Sys.time(),": Reloaded aggregate file from ", agg_path) +} else { + message(Sys.time(), ": Aggregating ", panel) + set.seed(1) + agg <- AggregateFlowFrames(file.path(preprocessed_path, + paste0(training_set, ".fcs")), + cTotal = 50000*length(training_set), + channels = colnames(ff)[1:32]) + write.FCS(agg, agg_path) + message(Sys.time(), ": Aggregating finished") +} + +# Train the FlowSOM model ------------------------------------------------------ + +if(!recompute & file.exists(fsom_path)){ + fsom <- readRDS(fsom_path) + message("Reloaded previously saved FlowSOM model from ", + fsom_path, + "\n Set recompute = TRUE to recompute.") +} else { + + n_meta <- 40 + t_fsom <- system.time( + fsom <- FlowSOM(agg, + colsToUse = channels_to_use, + xdim = 12, ydim = 12, + scale = FALSE, + nClus = n_meta, + seed = 1) + ) + + # Remove fluorochrome and channel + fsom$prettyColnames <- gsub(" [^ ]* <[^ ]*>$", "", fsom$prettyColnames) + # Remove channel if no fluorochrome was specified + fsom$prettyColnames <- gsub(" <[^ ]*>$", "", fsom$prettyColnames) + fsom$time <- t_fsom + + saveRDS(fsom, fsom_path) +} + +# Map all samples onto the model ----------------------------------------------- + +recompute <- TRUE + +for(i_start in seq(1, nrow(demographics), by = 100)){ + i_end <- min(i_start+99, nrow(demographics)) + message(Sys.time(),": ", i_start,"-",i_end) + + output_file <- gsub(".RDS$", paste0("_", i_start,"-", i_end, ".RDS"), output_path) + if(!recompute & file.exists(output_file)){ + message("Skipping previously saved features.", + "\n Set recompute = TRUE to recompute.") + } else { + files <- file.path(preprocessed_path, paste0(demographics$`FlowJo ID`[i_start:i_end], ".fcs")) + exists <- file.exists(files) + features <- GetFeatures(fsom, + files = files[exists], + level = c("clusters", "metaclusters"), + type = c("counts", "percentages", "MFIs"), + MFI = channels_MFI, + filenames = demographics$`FlowJo ID`[i_start:i_end][exists], + silent = FALSE) + + saveRDS(features, output_file) + } +} + +# Normalisation BDC panel ------------------------------------------------------ + +library(CytoNorm) + +# Training +panel <- "BDC" + +QC_samples <- demographics[demographics$Group == "QC",] +QC_files <- paste0(QC_samples$`FlowJo ID`,".fcs") + +batch <- QC_samples$`Run#` %in% c(5:19) +batch <- as.character(1 + batch) +normalisation_path <- file.path(base_dir, paste0(date, "_", panel, "_normalized")) + +nQ <- 50 +if(file.exists(file.path(normalisation_path, "CytoNorm_model_BDC.RDS"))){ + norm_model <- readRDS(file.path(normalisation_path, "CytoNorm_model_BDC.RDS")) +} else { + norm_model <- CytoNorm::CytoNorm.train(files = file.path(preprocessed_path, QC_files), + labels = batch, + channels = c(channels_to_use, channels_MFI), + transformList = NULL, + outputDir = normalisation_path, + FlowSOM.params = list(nCells = 1000000, + xdim = 12, ydim = 12, + nClus = 10, + channels = channels_to_use), + normMethod.train = QuantileNorm.train, + normParams = list(quantileValues = ((1:nQ)/nQ)[-nQ], + goal = "1", + limit = c(-1,4)), + seed = 1, + clean = TRUE, + plot = FALSE, + verbose = TRUE) + saveRDS(norm_model, + file.path(normalisation_path, "CytoNorm_model_BDC.RDS")) +} + +# Normalising +for(i_start in seq(1, nrow(demographics), by = 100)){ + i_end <- min(i_start+99, nrow(demographics)) + message(Sys.time(),": ", i_start,"-",i_end) + + files <- file.path(preprocessed_path, paste0(demographics$`FlowJo ID`[i_start:i_end], ".fcs")) + exists <- file.exists(files) + run <- demographics$`Run#`[i_start:i_end][exists] + + CytoNorm::CytoNorm.normalize(model = norm_model, + files = files[exists], + labels = as.character(1+(run %in% c(5:19))), + transformList = NULL, + transformList.reverse = NULL, + outputDir = normalisation_path, + prefix = "", + normMethod.normalize = QuantileNorm.normalize, + clean = TRUE, + verbose = TRUE) +} + +# Map all normalised samples onto the model + +for(i_start in seq(1, nrow(demographics), by = 100)){ + i_end <- min(i_start+99, nrow(demographics)) + message(Sys.time(),": ", i_start,"-",i_end) + + output_file <- gsub(".RDS$", paste0("_normalized_", i_start,"-", i_end, ".RDS"), output_path) + if(!recompute & file.exists(output_file)){ + message("Skipping previously saved features.", + "\n Set recompute = TRUE to recompute.") + } else { + files <- file.path(normalisation_path, paste0(demographics$`FlowJo ID`[i_start:i_end], ".fcs")) + exists <- file.exists(files) + features <- GetFeatures(fsom, + files = files[exists], + level = c("clusters", "metaclusters"), + type = c("counts", "percentages", "MFIs"), + MFI = channels_MFI, + filenames = demographics$`FlowJo ID`[i_start:i_end][exists], + silent = FALSE) + + saveRDS(features, output_file) + } +} diff --git a/README.md b/README.md index e7a9e25..a48fa99 100644 --- a/README.md +++ b/README.md @@ -1,2 +1,6 @@ # Liechti_et_al_TwinStudy Code used to generate the results described in Liechti et al. + +210708_loadDemographics.R loads the metadata of the fcs files. +210708_originalFlowSOMmodels.R uses this information to built FlowSOM models. +210708_figures.R creates all resulting figures, also using the helper function saved in 210708_compareSubset.R \ No newline at end of file diff --git a/schematic_overview.pdf b/schematic_overview.pdf new file mode 100644 index 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