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File Version Author Date Message
html d538aa2 Ziang Zhang 2026-07-28 Build site: reordered Figures 6 / S6 / S3 and the new Figure 7b page
html 029b0ae Ziang Zhang 2026-07-28 Build site.
Rmd 0f5b5da Ziang Zhang 2026-07-28 Align all figure captions with captions_20260728_final.docx
html 3fc3789 Ziang Zhang 2026-07-27 Republish all 24 pages
html 1390a03 Ziang Zhang 2026-07-27 Republish all 24 pages
html adaef21 Ziang Zhang 2026-07-27 Build site: panel fixes and PDF-derived assets
html 5b19858 Ziang Zhang 2026-07-27 Build site.
Rmd 91ee059 Ziang Zhang 2026-07-26 Select each panel’s code block by name, not by line number
html 91ee059 Ziang Zhang 2026-07-26 Select each panel’s code block by name, not by line number
Rmd 620afca Ziang Zhang 2026-07-24 Align Fig3/4/5 section titles with current numbers; reletter Fig4 to a-e
html 620afca Ziang Zhang 2026-07-24 Align Fig3/4/5 section titles with current numbers; reletter Fig4 to a-e
Rmd 37db4e6 Ziang Zhang 2026-07-18 Fig 5c: drop left expression heatmap, keep gene-score heatmap only
html 37db4e6 Ziang Zhang 2026-07-18 Fig 5c: drop left expression heatmap, keep gene-score heatmap only
Rmd a18012d Ziang Zhang 2026-07-16 Fig 5c: clip centered-expression color scale to [-1, 1]
html a18012d Ziang Zhang 2026-07-16 Fig 5c: clip centered-expression color scale to [-1, 1]
Rmd 3577da1 Ziang Zhang 2026-07-16 Fig 5c: center-only (no standardization), left panel as heatmap
html 3577da1 Ziang Zhang 2026-07-16 Fig 5c: center-only (no standardization), left panel as heatmap
Rmd 2873ad2 Ziang Zhang 2026-07-16 Unify Fig 3/4/5 panel filenames with new figure numbers
html 2873ad2 Ziang Zhang 2026-07-16 Unify Fig 3/4/5 panel filenames with new figure numbers
Rmd b197499 Ziang Zhang 2026-07-16 Restructure main figures (renumber + split Figure 1)
html b197499 Ziang Zhang 2026-07-16 Restructure main figures (renumber + split Figure 1)

All panels are produced by script/Figure5.R. The code below is shown for reference (not re-executed on this page); the images are its pre-rendered output.

Setup

Data loading, shared across all panels below.

# Figure 5. GPs and tissue.
#
# Panels produced. NOTE the renumbering: this figure's published counterpart is
# figures/Previous/bits/Figure *4* (4a-4e), not "Figure 5" -- there is no
# Figure 5 directory there. Full caption text:
# ../immgen-t-factors/figures/Figure_Organ/Figure_Organ_caption.md.
#   5a  Max AUC (organ) vs max AUC (level-1 lineage) scatter, per GP.
#   5b  GP37+ rate by lineage, mammary gland vs. the same lineage elsewhere.
#   5c  Marker genes of the 7 organ-specific GPs: per-GP gene-score heatmap.
#       The published panel (4c) paired this heatmap with an across-organ
#       expression dotplot on its left; the dotplot half was dropped on
#       purpose, so 5c is half the width of 4c by design (see the caption in
#       analysis/Figure5.Rmd, which describes the heatmap only).
#   5d  As 5a, but organ AUC vs Level-2 (fine-grained sub-lineage/cluster)
#       AUC, with the 7 organ-specific GPs (red) and a contrasting
#       cluster-specific set (blue) highlighted.
#   5e  Alluvial diagram: organ of origin -> GP -> Level-2 cell type, for
#       GP+ cells of the 7 organ-specific GPs.
#
# Source: ported from Figure_Organ.R, which mixed these 5 panels with
# other exploratory analyses (extra AUC scatter variants, per-organ ROC
# curves, a broken/undefined-object "gp_decomposition.pdf" panel) that are
# dropped here since they don't correspond to a final figure panel.
#
# Required inputs (data/) -- see code/README.md's "Data provenance" table
# for the full picture:
#   L_pm_filtered.rds, F_pm_filtered.rds     [code/pipeline/01b_filter_cells.R]
#   igt1_96_..._ADTonly.Rds                  [primary input Seurat object]
#   shifted_log_counts_subset.rds            [gap, no producer script here]
#   level_1_AUC_list_figure_no_thymocytes_healthy.rds,
#   level_2_AUC_list_figure_no_thymocytes_healthy.rds,
#   organ_simplified_AUC_list_figure_no_thymocytes_healthy.rds
#     [code/pipeline/02_compute_auc.R]

library(ggplot2)
library(ggrepel)
library(patchwork)
library(dplyr)
library(tidyr)
library(purrr)
library(tibble)
library(Matrix)
library(viridis)
library(cowplot)
library(ggalluvial)

data_path <- "data/"
figure_path <- "figures/final-selected/Figure 5/"

# ============================================================
# Load data (healthy, non-thymocyte reference)
# ============================================================
level_1_AUC_list <- readRDS(paste0(
  data_path, "level_1_AUC_list_figure_no_thymocytes_healthy.rds"
))
level_2_AUC_list <- readRDS(paste0(
  data_path, "level_2_AUC_list_figure_no_thymocytes_healthy.rds"
))
organ_AUC_list <- readRDS(paste0(
  data_path, "organ_simplified_AUC_list_figure_no_thymocytes_healthy.rds"
))
# Read from the Seurat object directly, not the stale cached seurat_meta.rds
# (see code/R/setup_data.R for why).
seurat_meta <- readRDS(paste0(
  data_path, "igt1_96_withtotalvi20260206_clean_ADTonly.Rds"
))@meta.data
L_pm_filtered <- readRDS(paste0(data_path, "L_pm_filtered.rds"))
seurat_meta_filtered <- seurat_meta[rownames(L_pm_filtered), ]

# Rename K## to GP## for display consistency
colnames(L_pm_filtered) <- gsub("^K", "GP", colnames(L_pm_filtered))
colnames(level_1_AUC_list$auc) <- gsub("^K", "GP", colnames(level_1_AUC_list$auc))
colnames(level_2_AUC_list$auc) <- gsub("^K", "GP", colnames(level_2_AUC_list$auc))
colnames(level_2_AUC_list$threshold) <- gsub("^K", "GP", colnames(level_2_AUC_list$threshold))
colnames(organ_AUC_list$auc) <- gsub("^K", "GP", colnames(organ_AUC_list$auc))
colnames(organ_AUC_list$threshold) <- gsub("^K", "GP", colnames(organ_AUC_list$threshold))

# Restrict reference to healthy, non-thymocyte cells
seurat_meta_filtered_no_thymocytes_healthy <- seurat_meta_filtered %>%
  filter(annotation_level1 != "thymocyte", condition_broad == "healthy")

# The 7 organ-specific GPs highlighted throughout this figure (caption 5d/5e)
gps_of_interest <- c("GP3", "GP6", "GP11", "GP26", "GP29", "GP37", "GP177")

# Labels a highlighted point with its top categories above `threshold` AUC.
top_cats_label <- function(factor_name, auc_matrix, positive_mask, threshold = 0.85, n = 3) {
  vals <- auc_matrix[, factor_name]
  vals <- vals[positive_mask[, factor_name]]
  vals <- sort(vals[vals > threshold], decreasing = TRUE)
  cats <- names(vals)[seq_len(min(n, length(vals)))]
  if (length(cats) == 0) return(factor_name)
  paste0(factor_name, ":\n", paste(cats, collapse = "\n"))
}

(a) Max AUC: organ vs. lineage

# ============================================================
# 5a: Max AUC Organ vs Level-1
# ============================================================
level_1_small_count <- table(seurat_meta_filtered_no_thymocytes_healthy$annotation_level1)
level_1_small_count <- names(level_1_small_count[level_1_small_count < 1000])
level_1_AUC <- level_1_AUC_list$auc
level_1_AUC <- level_1_AUC[!rownames(level_1_AUC) %in% level_1_small_count, ]

organ_AUC <- organ_AUC_list$auc
organ_small_count <- table(seurat_meta_filtered_no_thymocytes_healthy$organ_simplified)
organ_small_count <- names(organ_small_count[organ_small_count < 100])
organ_AUC <- organ_AUC[!rownames(organ_AUC) %in% organ_small_count, ]

# Positivity masks: mean loading in category > overall mean -> high loading predicts membership
healthy_cells <- rownames(seurat_meta_filtered_no_thymocytes_healthy)
L_healthy <- L_pm_filtered[healthy_cells, ]
overall_mean <- colMeans(L_healthy, na.rm = TRUE)

level_1_cat_mean <- t(sapply(rownames(level_1_AUC), function(cat) {
  idx <- seurat_meta_filtered_no_thymocytes_healthy$annotation_level1 == cat
  colMeans(L_healthy[idx, , drop = FALSE], na.rm = TRUE)
}))
level_1_AUC_positive <- sweep(level_1_cat_mean, 2, overall_mean, "-") > 0

organ_cat_mean <- t(sapply(rownames(organ_AUC), function(cat) {
  idx <- seurat_meta_filtered_no_thymocytes_healthy$organ_simplified == cat
  colMeans(L_healthy[idx, , drop = FALSE], na.rm = TRUE)
}))
organ_AUC_positive <- sweep(organ_cat_mean, 2, overall_mean, "-") > 0

level_1_AUC_masked <- level_1_AUC
level_1_AUC_masked[!level_1_AUC_positive] <- NA
level_1_AUC_max <- apply(level_1_AUC_masked, 2, max, na.rm = TRUE)
level_1_AUC_max_name <- apply(level_1_AUC_masked, 2, function(x) rownames(level_1_AUC_masked)[which.max(x)])
o <- order(level_1_AUC_max, decreasing = TRUE)
table_level_1_AUC <- data.frame(
  Factor = colnames(level_1_AUC)[o], Max_AUC = level_1_AUC_max[o], Annotation = level_1_AUC_max_name[o]
)

organ_AUC_masked <- organ_AUC
organ_AUC_masked[!organ_AUC_positive] <- NA
organ_AUC_max <- apply(organ_AUC_masked, 2, max, na.rm = TRUE)
organ_AUC_max_name <- apply(organ_AUC_masked, 2, function(x) rownames(organ_AUC_masked)[which.max(x)])

max_AUC_df <- data.frame(
  Factor = table_level_1_AUC$Factor,
  annotation_Level1 = table_level_1_AUC$Annotation,
  annotation_Organ = organ_AUC_max_name[match(table_level_1_AUC$Factor, names(organ_AUC_max))],
  Max_AUC_Organ = organ_AUC_max[match(table_level_1_AUC$Factor, names(organ_AUC_max))],
  Max_AUC_Level1 = table_level_1_AUC$Max_AUC
)
df <- max_AUC_df %>% mutate(residual = Max_AUC_Level1 - Max_AUC_Organ, abs_res = abs(residual))

# Factors to highlight: AUC > 0.9 in at least one axis (organ or level-1)
highlighted_factors <- df %>%
  filter(is.finite(residual), Max_AUC_Organ > 0.9 | Max_AUC_Level1 > 0.9) %>%
  pull(Factor)

label_above <- df %>%
  filter(Factor %in% highlighted_factors, residual > 0) %>%
  mutate(nudge_x = -0.035, label_text = sapply(Factor, top_cats_label, auc_matrix = level_1_AUC, positive_mask = level_1_AUC_positive))
label_below <- df %>%
  filter(Factor %in% highlighted_factors, residual <= 0) %>%
  mutate(nudge_x = 0.035, label_text = sapply(Factor, top_cats_label, auc_matrix = organ_AUC, positive_mask = organ_AUC_positive))

p_5a <- ggplot(df, aes(Max_AUC_Organ, Max_AUC_Level1)) +
  geom_point(alpha = 0.3, size = 1.8) +
  geom_point(data = label_above, color = "#1f78b4", alpha = 0.8, size = 1.8) +
  geom_point(data = label_below, color = "#e31a1c", alpha = 0.8, size = 1.8) +
  geom_abline(slope = 1, intercept = 0, linetype = "dashed", color = "black") +
  coord_cartesian(xlim = c(0.46, 1.04), ylim = c(0.5, 1.02), expand = FALSE) +
  labs(x = "Max AUC (Organ Simplified)", y = "Max AUC (Level-1)", title = "Max AUC: Organ vs Level-1") +
  theme_minimal(base_size = 13) +
  geom_text_repel(
    seed = 42,
    data = label_above, aes(label = label_text), color = "#1f78b4", size = 2.5, lineheight = 0.85,
    direction = "y", nudge_x = label_above$nudge_x, segment.color = "#1f78b4",
    arrow = arrow(length = unit(0.008, "npc"), type = "closed", angle = 20),
    force = 3, force_pull = 0.1, box.padding = 0.4, point.padding = 0.15,
    max.time = 10, max.iter = 2e4, max.overlaps = 20, min.segment.length = 0.01, segment.alpha = 0.7
  ) +
  geom_text_repel(
    seed = 42,
    data = label_below, aes(label = label_text), color = "#e31a1c", size = 2.5, lineheight = 0.85,
    direction = "y", nudge_x = label_below$nudge_x, segment.color = "#e31a1c",
    arrow = arrow(length = unit(0.008, "npc"), type = "closed", angle = 20),
    force = 3, force_pull = 0.1, box.padding = 0.4, point.padding = 0.15,
    max.time = 10, max.iter = 2e4, max.overlaps = 20, min.segment.length = 0.01, segment.alpha = 0.7
  )
ggsave(filename = paste0(figure_path, "5a.pdf"), plot = p_5a, width = 8, height = 8, dpi = 300)

Version Author Date
7102598 Ziang Zhang 2026-07-27
6c1a613 Ziang Zhang 2026-07-27
bf7afcf Ziang Zhang 2026-07-27
ea3ecc2 Ziang Zhang 2026-07-27
2873ad2 Ziang Zhang 2026-07-16

Fig. 5a. Per-GP maximum AUC for predicting organ of origin (x-axis) versus lineage (y-axis); each point is one GP and the dashed line marks equal performance. Categories with too few cells are dropped (lineage < 1,000 cells, organ < 100 cells). GPs reaching AUC > 0.9 on either axis are highlighted – red below the diagonal (better at organ; organ-specific) labeled with their top organs, blue above the diagonal (better at lineage) labeled with their top lineages (up to three categories each, AUC > 0.85).

(b) GP37+ rate in mammary gland vs. elsewhere

# ============================================================
# 5b: GP37+ rate by lineage, mammary gland vs. elsewhere
# ============================================================
plot_gp_threshold_group_activation_rate <- function(
  gp, organ, threshold, loading_mat, organ_info, group_info,
  group_label = "Level-2", base_size = 13, min_in_organ = 10,
  group_colors = ZemmourLib::immgent_colors$level2, fallback_group_color = "grey60",
  reference = c("not_in_group", "not_in_organ")
) {
  reference <- match.arg(reference)
  if (!gp %in% colnames(loading_mat)) stop(sprintf("GP '%s' not found in loading matrix.", gp))
  if (!organ %in% organ_info) stop(sprintf("Organ '%s' not found in organ_info.", organ))

  loading <- loading_mat[, gp]
  keep <- !(is.na(loading) | is.na(organ_info) | is.na(group_info))
  loading <- loading[keep]
  organ_info <- organ_info[keep]
  group_info <- as.character(group_info[keep])

  in_organ <- organ_info == organ
  positive <- loading > threshold
  group_levels <- sort(unique(group_info))

  rate_df <- data.frame(
    group = group_levels,
    n_in_organ = vapply(group_levels, function(l) sum(group_info == l & in_organ), integer(1)),
    n_pos_in_organ = vapply(group_levels, function(l) sum(group_info == l & in_organ & positive), integer(1))
  )

  if (reference == "not_in_group") {
    rate_df$n_ref <- vapply(group_levels, function(l) sum(group_info != l & in_organ), integer(1))
    rate_df$n_pos_ref <- vapply(group_levels, function(l) sum(group_info != l & in_organ & positive), integer(1))
    ref_label <- "Not in group (same organ)"
    title_vs <- sprintf("%s vs. same-organ non-group", organ)
  } else {
    rate_df$n_ref <- vapply(group_levels, function(l) sum(group_info == l & !in_organ), integer(1))
    rate_df$n_pos_ref <- vapply(group_levels, function(l) sum(group_info == l & !in_organ & positive), integer(1))
    ref_label <- "Not in organ (same group)"
    title_vs <- sprintf("%s vs. same-group non-organ", organ)
  }

  rate_df$rate_in_organ <- rate_df$n_pos_in_organ / rate_df$n_in_organ
  rate_df$rate_ref <- rate_df$n_pos_ref / rate_df$n_ref
  rate_df <- rate_df[rate_df$n_in_organ >= min_in_organ, , drop = FALSE]
  if (nrow(rate_df) == 0) stop(sprintf("No %s type has >= %d cells in '%s'.", group_label, min_in_organ, organ))

  long_df <- data.frame(
    group = rep(rate_df$group, 2),
    type = factor(rep(c("In organ", ref_label), each = nrow(rate_df)), levels = c("In organ", ref_label)),
    rate = c(rate_df$rate_in_organ, rate_df$rate_ref)
  )
  level_order <- rate_df$group[order(rate_df$rate_in_organ, decreasing = TRUE)]
  long_df$group <- factor(long_df$group, levels = level_order)

  fill_values <- group_colors[as.character(level_order)]
  missing_colors <- is.na(fill_values)
  if (any(missing_colors)) {
    fill_values[missing_colors] <- fallback_group_color
    warning(sprintf(
      "%s annotations missing from group_colors and colored %s: %s",
      group_label, fallback_group_color, paste(level_order[missing_colors], collapse = ", ")
    ))
  }
  alpha_vals <- c(1, 0.35)
  names(alpha_vals) <- c("In organ", ref_label)

  ggplot(long_df, aes(x = group, y = rate, fill = group, alpha = type)) +
    geom_col(position = position_dodge(width = 0.8), width = 0.75, color = "grey35", linewidth = 0.15) +
    scale_fill_manual(values = fill_values, guide = "none") +
    scale_alpha_manual(values = alpha_vals, guide = guide_legend(override.aes = list(fill = "grey40"))) +
    labs(
      x = sprintf("%s annotation", group_label),
      y = sprintf("Proportion of cells with %s > %.3g", gp, threshold),
      alpha = NULL,
      title = sprintf("%s+ rate by %s: %s", gp, group_label, title_vs),
      subtitle = sprintf("threshold = %.3g; %s types with < %d cells in %s dropped", threshold, group_label, min_in_organ, organ)
    ) +
    theme_minimal(base_size = base_size) +
    theme(axis.text.x = element_text(angle = 45, hjust = 1), legend.position = "top")
}

p_5b <- plot_gp_threshold_group_activation_rate(
  gp = "GP37",
  organ = "mammary gland",
  threshold = organ_AUC_list$threshold["mammary gland", "GP37"],
  min_in_organ = 100,
  loading_mat = L_pm_filtered[rownames(seurat_meta_filtered_no_thymocytes_healthy), ],
  organ_info = seurat_meta_filtered_no_thymocytes_healthy$organ_simplified,
  group_info = seurat_meta_filtered_no_thymocytes_healthy$annotation_level1,
  group_label = "Level-1",
  group_colors = ZemmourLib::immgent_colors$level1,
  reference = "not_in_organ"
)
ggsave(filename = paste0(figure_path, "5b.pdf"), plot = p_5b, width = 8, height = 5, dpi = 300)

Version Author Date
7102598 Ziang Zhang 2026-07-27
6c1a613 Ziang Zhang 2026-07-27
bf7afcf Ziang Zhang 2026-07-27
ea3ecc2 Ziang Zhang 2026-07-27
2873ad2 Ziang Zhang 2026-07-16

Fig. 5b. Proportion of GP37-active cells in each lineage in the mammary gland (solid bars) compared with the same lineage in all other organs (faded bars). A cell is GP37-active (GP37+) if its GP37 loading exceeds the organ-specific optimal threshold from the AUC analysis. Bars are colored by lineage and ordered by in-organ rate; lineages with fewer than 100 mammary-gland cells are omitted.

(c) Marker genes of the 7 organ-specific GPs

# ============================================================
# 5c: organ marker genes - per-GP gene-score heatmap
# ============================================================
F_pm_filtered <- readRDS(paste0(data_path, "F_pm_filtered.rds"))
colnames(F_pm_filtered) <- gsub("^F", "GP", colnames(F_pm_filtered))
D_scale <- diag(1 / apply(F_pm_filtered, 2, function(x) max(abs(x), na.rm = TRUE)))
F_pm_scaled <- F_pm_filtered %*% D_scale
colnames(F_pm_scaled) <- colnames(F_pm_filtered)

n_top_genes <- 20
min_loading <- 0.25
F_sub <- F_pm_scaled[, gps_of_interest, drop = FALSE]
selected_genes <- lapply(gps_of_interest, function(gp) {
  vals <- F_sub[, gp]
  names(sort(vals[vals > min_loading], decreasing = TRUE))[seq_len(min(n_top_genes, sum(vals > min_loading)))]
})
selected_genes <- unique(unlist(selected_genes))

# Diagonal gene ordering by dominant GP (highest loading)
GP_orders <- c("GP37", "GP26", "GP6", "GP177", "GP3", "GP29", "GP11")
dominant_gp <- apply(F_sub[selected_genes, , drop = FALSE], 1, function(x) GP_orders[which.max(x[GP_orders])])
dominant_loading <- mapply(function(g, gp) F_sub[g, gp], selected_genes, dominant_gp)
gene_order_df <- data.frame(Gene = selected_genes, dominant_gp = factor(dominant_gp, levels = GP_orders), loading = dominant_loading, stringsAsFactors = FALSE)
gene_order_df <- gene_order_df[order(gene_order_df$dominant_gp, -gene_order_df$loading), ]
heatmap_gene_order <- gene_order_df$Gene

plot_df_hm <- as.data.frame(F_sub[heatmap_gene_order, , drop = FALSE])
plot_df_hm$Gene <- rownames(plot_df_hm)
plot_df_hm <- tidyr::pivot_longer(plot_df_hm, cols = -Gene, names_to = "GP", values_to = "Loading")
plot_df_hm$GP <- factor(plot_df_hm$GP, levels = GP_orders)
plot_df_hm$Gene <- factor(plot_df_hm$Gene, levels = rev(heatmap_gene_order))
limit_hm <- max(abs(plot_df_hm$Loading), na.rm = TRUE)

p_gene_heatmap <- ggplot(plot_df_hm, aes(x = GP, y = Gene, fill = Loading)) +
  geom_tile() +
  scale_fill_gradient2(low = "steelblue", mid = "white", high = "firebrick", midpoint = 0, limits = c(-limit_hm, limit_hm), name = "Loading") +
  labs(title = "Top positive genes per organ GP", x = NULL, y = NULL) +
  theme_minimal(base_size = 9) +
  theme(axis.text.x = element_text(angle = 45, hjust = 1, size = 9), axis.text.y = element_text(size = 8), panel.grid = element_blank(), plot.title = element_text(face = "bold", size = 11))

pdf(paste0(figure_path, "5c.pdf"), width = 5, height = 16, useDingbats = FALSE)
print(p_gene_heatmap)
dev.off()

Version Author Date
7102598 Ziang Zhang 2026-07-27
6c1a613 Ziang Zhang 2026-07-27
bf7afcf Ziang Zhang 2026-07-27
ea3ecc2 Ziang Zhang 2026-07-27
37db4e6 Ziang Zhang 2026-07-18
a18012d Ziang Zhang 2026-07-16
3577da1 Ziang Zhang 2026-07-16
2873ad2 Ziang Zhang 2026-07-16

Fig. 5c. Marker genes of the seven organ-specific GPs. Genes are the union of the top 20 positively scoring genes per GP (score > 0.25 on the max-abs-scaled gene-score matrix), ordered diagonally by each gene’s dominant GP and then by descending score. Heatmap of the per-GP scaled gene scores for these genes across the seven GPs, centered at zero (blue, negative; red, positive).

(d) Max AUC: organ vs. cluster

# ============================================================
# 5d prep: Max AUC Organ vs Level-2
# ============================================================
level_2_AUC <- level_2_AUC_list$auc
level_2_small_count <- table(seurat_meta_filtered_no_thymocytes_healthy$annotation_level2)
level_2_small_count <- names(level_2_small_count[level_2_small_count < 100])
level_2_AUC <- level_2_AUC[!rownames(level_2_AUC) %in% level_2_small_count, ]

level_2_cat_mean <- t(sapply(rownames(level_2_AUC), function(cat) {
  idx <- seurat_meta_filtered_no_thymocytes_healthy$annotation_level2 == cat
  colMeans(L_healthy[idx, , drop = FALSE], na.rm = TRUE)
}))
level_2_AUC_positive <- sweep(level_2_cat_mean, 2, overall_mean, "-") > 0

# 5d/5e reuse `organ_AUC_max_name`, but recomputed against the Level-2
# category-count filter to match the original script's exact numbers.
organ_AUC_masked_l2 <- organ_AUC
organ_AUC_positive_l2 <- sweep(
  t(sapply(rownames(organ_AUC), function(cat) {
    idx <- seurat_meta_filtered_no_thymocytes_healthy$organ_simplified == cat
    colMeans(L_healthy[idx, , drop = FALSE], na.rm = TRUE)
  })),
  2, overall_mean, "-"
) > 0
organ_AUC_masked_l2[!organ_AUC_positive_l2] <- NA
organ_AUC_max <- apply(organ_AUC_masked_l2, 2, max, na.rm = TRUE)
organ_AUC_max_name <- apply(organ_AUC_masked_l2, 2, function(x) rownames(organ_AUC_masked_l2)[which.max(x)])

# 5d needs its OWN max-AUC table -- it must not reuse 5a's `df`, whose
# Max_AUC_Level1 column holds the Level-1 maxima. The original Figure_Organ.R
# rebuilds `max_AUC_df`/`df` at this point from `table_level_2_AUC` (storing
# the Level-2 maxima in a column it still calls `Max_AUC_Level1` -- a
# misleading name we drop here in favour of `Max_AUC_Level2`). Reusing 5a's
# `df` silently plots Level-1 AUC on this panel's "Max AUC (Level-2)" axis.
level_2_AUC_masked <- level_2_AUC
level_2_AUC_masked[!level_2_AUC_positive] <- NA
level_2_AUC_max <- apply(level_2_AUC_masked, 2, max, na.rm = TRUE)
level_2_AUC_max_name <- apply(level_2_AUC_masked, 2, function(x) {
  rownames(level_2_AUC_masked)[which.max(x)]
})
o_l2 <- order(level_2_AUC_max, decreasing = TRUE)
table_level_2_AUC <- data.frame(
  Factor = colnames(level_2_AUC)[o_l2],
  Max_AUC = level_2_AUC_max[o_l2],
  Annotation = level_2_AUC_max_name[o_l2]
)
df_l2 <- data.frame(
  Factor = table_level_2_AUC$Factor,
  annotation_Level2 = table_level_2_AUC$Annotation,
  annotation_Organ = organ_AUC_max_name[match(table_level_2_AUC$Factor, names(organ_AUC_max))],
  Max_AUC_Organ = organ_AUC_max[match(table_level_2_AUC$Factor, names(organ_AUC_max))],
  Max_AUC_Level2 = table_level_2_AUC$Max_AUC
) %>%
  mutate(residual = Max_AUC_Level2 - Max_AUC_Organ, abs_res = abs(residual))

# ============================================================
# 5d: Max AUC Organ vs Level-2, 7 organ-specific GPs (red) vs.
#     contrasting cluster-specific GPs (blue) highlighted
# ============================================================
seven_gp_df <- df_l2 |>
  dplyr::filter(Factor %in% gps_of_interest) |>
  dplyr::mutate(label_text = sapply(Factor, top_cats_label, auc_matrix = level_2_AUC, positive_mask = level_2_AUC_positive, threshold = 0.9, n = 3))

top_left_gps <- c("GP14", "GP36", "GP16", "GP151", "GP21", "GP122", "GP2", "GP171", "GP5", "GP13")
top_left_df <- df_l2 |>
  dplyr::filter(Factor %in% top_left_gps) |>
  dplyr::mutate(label_text = sapply(Factor, top_cats_label, auc_matrix = level_2_AUC, positive_mask = level_2_AUC_positive, threshold = 0.9, n = 3))

p_5d <- ggplot(df_l2, aes(Max_AUC_Organ, Max_AUC_Level2)) +
  geom_point(alpha = 0.2, size = 1.5, color = "grey60") +
  geom_point(data = top_left_df, color = "#1f78b4", size = 2.2, alpha = 0.9) +
  geom_text_repel(
    seed = 42,
    data = top_left_df, aes(label = label_text), color = "#1f78b4", lineheight = 0.85, size = 2.5,
    direction = "y", nudge_x = -0.1, segment.color = "#1f78b4",
    arrow = arrow(length = unit(0.008, "npc"), type = "closed", angle = 20),
    force = 4, force_pull = 0.05, box.padding = 0.5, point.padding = 0.15,
    max.time = 10, max.iter = 2e4, max.overlaps = 30, min.segment.length = 0.01, segment.alpha = 0.7
  ) +
  geom_point(data = seven_gp_df, color = "#e31a1c", size = 2.2, alpha = 0.9) +
  geom_text_repel(
    seed = 42,
    data = seven_gp_df, aes(label = label_text), color = "#e31a1c", size = 2.5, lineheight = 0.85,
    direction = "y", nudge_x = 0.18, xlim = c(1.0, NA), segment.color = "#e31a1c",
    arrow = arrow(length = unit(0.008, "npc"), type = "closed", angle = 20),
    force = 6, force_pull = 0.02, box.padding = 0.6, point.padding = 0.15,
    max.time = 10, max.iter = 2e4, max.overlaps = 30, min.segment.length = 0.01, segment.alpha = 0.7
  ) +
  geom_abline(slope = 1, intercept = 0, linetype = "dashed", color = "black") +
  coord_cartesian(xlim = c(0.46, 1.04), ylim = c(0.5, 1.02), expand = FALSE, clip = "off") +
  labs(x = "Max AUC (Organ Simplified)", y = "Max AUC (Level-2)", title = "Max AUC: Organ vs Level-2 - organ-specific GPs") +
  theme_minimal(base_size = 13) +
  theme(plot.margin = margin(10, 80, 10, 80))
ggsave(filename = paste0(figure_path, "5d.pdf"), plot = p_5d, width = 8, height = 8, dpi = 300)

Version Author Date
7102598 Ziang Zhang 2026-07-27
6c1a613 Ziang Zhang 2026-07-27
bf7afcf Ziang Zhang 2026-07-27
ea3ecc2 Ziang Zhang 2026-07-27
2873ad2 Ziang Zhang 2026-07-16

Fig. 5d. As in (a), but predictive performance is measured against level 2 clusters (annotation_level2, from ImmgenT-cosmo; y-axis, clusters with < 100 cells dropped). All GPs are shown in grey; the seven organ-specific GPs from (a) – GP3, GP6, GP11, GP26, GP29, GP37, GP177 – are highlighted in red and labeled with their best clusters, and a contrasting set of cluster-specific GPs (high cluster AUC, low organ AUC) in blue; the dashed line marks equal performance.

(e) Organ, GP, and cluster alluvial diagram

# ============================================================
# 5e: alluvial, organ -> GP -> Level-2, for GP+ cells of the
#     7 organ-specific GPs
# ============================================================
best_organ_per_gp <- organ_AUC_max_name[gps_of_interest]
gp_thresholds <- mapply(function(gp, organ) organ_AUC_list$threshold[organ, gp], gps_of_interest, best_organ_per_gp)
names(gp_thresholds) <- gps_of_interest

n_cap_gp <- 300
set.seed(42)
alluvial_rows <- lapply(gps_of_interest, function(gp) {
  positive_idx <- L_healthy[, gp] > gp_thresholds[gp]
  meta_pos <- seurat_meta_filtered_no_thymocytes_healthy[positive_idx, ]
  d <- data.frame(gp_program = gp, organ = meta_pos$organ_simplified, level2 = meta_pos$annotation_level2, stringsAsFactors = FALSE)
  if (nrow(d) > n_cap_gp) d <- dplyr::slice_sample(d, n = n_cap_gp)
  d
})

count_df <- do.call(rbind, alluvial_rows) |>
  dplyr::count(organ, gp_program, level2, name = "n") |>
  dplyr::filter(!is.na(organ), !is.na(level2), n >= 5)

organ_order <- count_df |> dplyr::summarise(total = sum(n), .by = organ) |> dplyr::arrange(dplyr::desc(total)) |> dplyr::pull(organ)
level2_order <- count_df |> dplyr::summarise(total = sum(n), .by = level2) |> dplyr::arrange(dplyr::desc(total)) |> dplyr::pull(level2)

count_df <- count_df |>
  dplyr::mutate(
    organ = factor(organ, levels = rev(organ_order)),
    gp_program = factor(gp_program, levels = rev(gps_of_interest)),
    level2 = factor(level2, levels = rev(level2_order))
  )

gp_colors <- ZemmourLib::immgent_colors$organ_simplified[unname(best_organ_per_gp)]
gp_colors[is.na(gp_colors)] <- "grey60"
names(gp_colors) <- gps_of_interest

p_5e <- ggplot(count_df, aes(axis1 = organ, axis2 = gp_program, axis3 = level2, y = n)) +
  ggalluvial::geom_alluvium(aes(fill = gp_program), width = 1 / 4, alpha = 0.6, knot.pos = 0.4) +
  ggalluvial::geom_stratum(width = 1 / 4, fill = "grey92", color = "grey50", linewidth = 0.3) +
  ggplot2::geom_text(stat = ggalluvial::StatStratum, aes(label = after_stat(stratum)), size = 3, angle = 90) +
  scale_fill_manual(values = gp_colors, guide = "none") +
  scale_x_discrete(limits = c("Organ", "GP", "Level-2"), expand = c(0.12, 0.12)) +
  labs(y = "Number of GP+ cells", title = "GP+ cells: organ origin and cell type") +
  theme_minimal(base_size = 12) +
  theme(panel.grid = element_blank(), axis.text.y = element_blank(), axis.ticks = element_blank()) +
  coord_flip()
ggsave(filename = paste0(figure_path, "5e.pdf"), plot = p_5e, width = 20, height = 10, dpi = 300)

Version Author Date
7102598 Ziang Zhang 2026-07-27
6c1a613 Ziang Zhang 2026-07-27
bf7afcf Ziang Zhang 2026-07-27
ea3ecc2 Ziang Zhang 2026-07-27
2873ad2 Ziang Zhang 2026-07-16

Fig. 5e. Alluvial plot showing how tissue-specific GP activity is distributed across clusters. For each of the seven organ-specific GPs and each organ, GP-active cells (loading above that GP’s best-organ threshold) are traced from their organ of origin through the GP to their level 2 cluster annotation. Flows are colored by GP (using each GP’s best-predicted organ color), and width is proportional to the number of cells shown, after subsampling to <=300 cells per GP; flows representing fewer than 5 cells are dropped, and a cell may appear under more than one GP.


sessionInfo()
R version 4.5.1 (2025-06-13)
Platform: aarch64-apple-darwin20
Running under: macOS Sequoia 15.6.1

Matrix products: default
BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.5-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.1

locale:
[1] en_CA/en_CA/en_CA/C/en_CA/en_CA

time zone: America/Chicago
tzcode source: internal

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

loaded via a namespace (and not attached):
 [1] vctrs_0.7.3     cli_3.6.6       knitr_1.50      rlang_1.2.0    
 [5] xfun_0.55       stringi_1.8.7   otel_0.2.0      promises_1.5.0 
 [9] jsonlite_2.0.0  workflowr_1.7.2 glue_1.8.1      rprojroot_2.1.1
[13] git2r_0.36.2    htmltools_0.5.9 httpuv_1.6.16   sass_0.4.10    
[17] rmarkdown_2.30  evaluate_1.0.5  jquerylib_0.1.4 tibble_3.3.0   
[21] fastmap_1.2.0   yaml_2.3.12     lifecycle_1.0.5 whisker_0.4.1  
[25] stringr_1.6.0   compiler_4.5.1  fs_1.6.6        Rcpp_1.1.1-1.1 
[29] pkgconfig_2.0.3 later_1.4.4     digest_0.6.39   R6_2.6.1       
[33] pillar_1.11.1   magrittr_2.0.5  bslib_0.9.0     tools_4.5.1    
[37] cachem_1.1.0