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scripts/quantify_regions.R

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+# Calling Cards Region Quantification
+# Quantifies enrichment of insertions/hops in genomic regions
+#
+# This script:
+# 1. Counts insertions overlapping each genomic region (experiment and background)
+# 2. Calculates enrichment scores
+# 3. Computes Poisson and hypergeometric p-values
+#
+# Works with any data in BED3+ format (chr, start, end, ...)
+# For calling cards: each insertion is counted once regardless of depth
+#
+# COORDINATE SYSTEMS:
+# - Input BED files are assumed to be 0-indexed, half-open [start, end)
+# - GenomicRanges uses 1-indexed, closed [start, end]
+# - Conversion: GR_start = BED_start + 1, GR_end = BED_end
+#
+# Usage:
+#   Rscript quantify_regions.R
+
+library(tidyverse)
+library(GenomicRanges)
+
+# Statistical Functions ---------------------------------------------------
+
+#' Calculate enrichment (calling cards effect)
+#'
+#' @param total_background_hops Total number of hops in background (scalar or vector)
+#' @param total_experiment_hops Total number of hops in experiment (scalar or vector)
+#' @param background_hops Number of hops in background per region (vector)
+#' @param experiment_hops Number of hops in experiment per region (vector)
+#' @param pseudocount Pseudocount to avoid division by zero (default: 0.1)
+#' @return Enrichment values
+calculate_enrichment <- function(total_background_hops,
+                                 total_experiment_hops,
+                                 background_hops,
+                                 experiment_hops,
+                                 pseudocount = 0.1) {
+
+  # Input validation
+  if (!all(is.numeric(c(total_background_hops, total_experiment_hops,
+                        background_hops, experiment_hops)))) {
+    stop("All inputs must be numeric")
+  }
+
+  # Get the length of the region vectors
+  n_regions <- length(background_hops)
+
+  # Ensure experiment_hops is same length as background_hops
+  if (length(experiment_hops) != n_regions) {
+    stop("background_hops and experiment_hops must be the same length")
+  }
+
+  # Recycle scalar totals to match region length if needed
+  if (length(total_background_hops) == 1) {
+    total_background_hops <- rep(total_background_hops, n_regions)
+  }
+  if (length(total_experiment_hops) == 1) {
+    total_experiment_hops <- rep(total_experiment_hops, n_regions)
+  }
+
+  # Now check all are same length
+  if (length(total_background_hops) != n_regions ||
+      length(total_experiment_hops) != n_regions) {
+    stop("All input vectors must be the same length or scalars")
+  }
+
+  # Calculate enrichment
+  numerator <- experiment_hops / total_experiment_hops
+  denominator <- (background_hops + pseudocount) / total_background_hops
+  enrichment <- numerator / denominator
+
+  # Check for invalid values
+  if (any(enrichment < 0, na.rm = TRUE)) {
+    stop("Enrichment values must be non-negative")
+  }
+  if (any(is.na(enrichment))) {
+    stop("Enrichment values must not be NA")
+  }
+  if (any(is.infinite(enrichment))) {
+    stop("Enrichment values must not be infinite")
+  }
+
+  return(enrichment)
+}
+
+
+#' Calculate Poisson p-values
+#'
+#' @param total_background_hops Total number of hops in background (scalar or vector)
+#' @param total_experiment_hops Total number of hops in experiment (scalar or vector)
+#' @param background_hops Number of hops in background per region (vector)
+#' @param experiment_hops Number of hops in experiment per region (vector)
+#' @param pseudocount Pseudocount for lambda calculation (default: 0.1)
+#' @param ... additional arguments to `ppois`. note that lower tail is set to FALSE
+#'   already
+#' @return Poisson p-values
+calculate_poisson_pval <- function(total_background_hops,
+                                   total_experiment_hops,
+                                   background_hops,
+                                   experiment_hops,
+                                   pseudocount = 0.1,
+                                   ...) {
+
+  # Input validation
+  if (!all(is.numeric(c(total_background_hops, total_experiment_hops,
+                        background_hops, experiment_hops)))) {
+    stop("All inputs must be numeric")
+  }
+
+  # Get the length of the region vectors
+  n_regions <- length(background_hops)
+
+  # Ensure experiment_hops is same length as background_hops
+  if (length(experiment_hops) != n_regions) {
+    stop("background_hops and experiment_hops must be the same length")
+  }
+
+  # Recycle scalar totals to match region length if needed
+  if (length(total_background_hops) == 1) {
+    total_background_hops <- rep(total_background_hops, n_regions)
+  }
+  if (length(total_experiment_hops) == 1) {
+    total_experiment_hops <- rep(total_experiment_hops, n_regions)
+  }
+
+  # Now check all are same length
+  if (length(total_background_hops) != n_regions ||
+      length(total_experiment_hops) != n_regions) {
+    stop("All input vectors must be the same length or scalars")
+  }
+
+  # Calculate hop ratio
+  hop_ratio <- total_experiment_hops / total_background_hops
+
+  # Calculate expected number of hops (mu/lambda parameter)
+  # Add pseudocount to avoid mu = 0
+  mu <- (background_hops + pseudocount) * hop_ratio
+
+  # Observed hops in experiment
+  x <- experiment_hops
+
+  # Calculate p-value: P(X >= x) = 1 - P(X < x) = 1 - P(X <= x-1)
+  # This is equivalent to: 1 - CDF(x) + PMF(x)
+  # Using the upper tail directly with lower.tail = FALSE
+  pval <- ppois(x - 1, lambda = mu, lower.tail = FALSE)
+
+  return(pval)
+}
+
+
+#' Calculate hypergeometric p-values
+#'
+#' @param total_background_hops Total number of hops in background (scalar or vector)
+#' @param total_experiment_hops Total number of hops in experiment (scalar or vector)
+#' @param background_hops Number of hops in background per region (vector)
+#' @param experiment_hops Number of hops in experiment per region (vector)
+#' @param ... additional arguments to phyper. Note that lower tail is set to
+#'   false already
+#' @return Hypergeometric p-values
+calculate_hypergeom_pval <- function(total_background_hops,
+                                     total_experiment_hops,
+                                     background_hops,
+                                     experiment_hops,
+                                     ...) {
+
+  # Input validation
+  if (!all(is.numeric(c(total_background_hops, total_experiment_hops,
+                        background_hops, experiment_hops)))) {
+    stop("All inputs must be numeric")
+  }
+
+  # Get the length of the region vectors
+  n_regions <- length(background_hops)
+
+  # Ensure experiment_hops is same length as background_hops
+  if (length(experiment_hops) != n_regions) {
+    stop("background_hops and experiment_hops must be the same length")
+  }
+
+  # Recycle scalar totals to match region length if needed
+  if (length(total_background_hops) == 1) {
+    total_background_hops <- rep(total_background_hops, n_regions)
+  }
+  if (length(total_experiment_hops) == 1) {
+    total_experiment_hops <- rep(total_experiment_hops, n_regions)
+  }
+
+  # Now check all are same length
+  if (length(total_background_hops) != n_regions ||
+      length(total_experiment_hops) != n_regions) {
+    stop("All input vectors must be the same length or scalars")
+  }
+
+  # Hypergeometric parameters
+  # M: total number of balls (total hops)
+  M <- total_background_hops + total_experiment_hops
+  # n: number of white balls (experiment hops)
+  n <- total_experiment_hops
+  # N: number of draws (hops in region)
+  N <- background_hops + experiment_hops
+  # x: number of white balls drawn (experiment hops in region) - 1 for upper tail
+  x <- experiment_hops - 1
+
+  # Handle edge cases
+  valid <- (M >= 1) & (N >= 1)
+  pval <- rep(1, length(M))
+
+  # Calculate p-value for valid cases: P(X >= x) = 1 - P(X <= x-1)
+  if (any(valid)) {
+    pval[valid] <- phyper(x[valid], n[valid], M[valid] - n[valid], N[valid],
+                          lower.tail = FALSE, ...)
+  }
+
+  return(pval)
+}
+
+# GRanges Conversion Functions --------------------------------------------
+
+#' Convert BED format data frame to GRanges
+#'
+#' Handles coordinate system conversion from 0-indexed half-open BED format
+#' to 1-indexed closed GenomicRanges format
+#'
+#' @param bed_df Data frame with chr, start, end columns in BED format (0-indexed, half-open)
+#' @param zero_indexed Logical, whether input is 0-indexed (default: TRUE)
+#' @return GRanges object
+bed_to_granges <- function(bed_df, zero_indexed = TRUE) {
+
+  if (!all(c("chr", "start", "end") %in% names(bed_df))) {
+    stop("bed_df must have columns: chr, start, end")
+  }
+
+  # Convert from 0-indexed half-open [start, end) to 1-indexed closed [start, end]
+  if (zero_indexed) {
+    gr_start <- bed_df$start + 1
+    gr_end <- bed_df$end
+  } else {
+    gr_start <- bed_df$start
+    gr_end <- bed_df$end
+  }
+
+  # Create GRanges object (strand-agnostic for calling cards)
+  gr <- GRanges(
+    seqnames = bed_df$chr,
+    ranges = IRanges(start = gr_start, end = gr_end),
+    strand = "*"
+  )
+
+  # Add any additional metadata columns
+  extra_cols <- setdiff(names(bed_df), c("chr", "start", "end", "strand"))
+  if (length(extra_cols) > 0) {
+    mcols(gr) <- bed_df[, extra_cols, drop = FALSE]
+  }
+
+  return(gr)
+}
+
+
+#' Deduplicate insertions in GRanges object
+#'
+#' For calling cards, if an insertion is found at the same coordinate,
+#' only one record is retained
+#'
+#' @param gr GRanges object
+#' @return Deduplicated GRanges object
+deduplicate_granges <- function(gr) {
+  # Find unique ranges (ignores strand and metadata)
+  unique_ranges <- !duplicated(granges(gr))
+  gr[unique_ranges]
+}
+
+
+#' Count overlaps between insertions and regions
+#'
+#' @param insertions_gr GRanges object with insertions
+#' @param regions_gr GRanges object with regions
+#' @param deduplicate Whether to deduplicate insertions (default: TRUE)
+#' @return Integer vector of overlap counts per region
+count_overlaps <- function(insertions_gr, regions_gr, deduplicate = TRUE) {
+
+  # Deduplicate if requested
+  if (deduplicate) {
+    n_before <- length(insertions_gr)
+    insertions_gr <- deduplicate_granges(insertions_gr)
+    n_after <- length(insertions_gr)
+    if (n_before != n_after) {
+      message("   Deduplicated: ", n_before, " -> ", n_after,
+              " (removed ", n_before - n_after, " duplicates)")
+    }
+  }
+
+  # Count overlaps per region
+  # countOverlaps returns an integer vector with one element per region
+  counts <- countOverlaps(regions_gr, insertions_gr)
+
+  return(counts)
+}
+
+
+# Main Analysis Function --------------------------------------------------
+
+#' Call peaks/quantify regions using calling cards approach
+#'
+#' @param experiment_gr GRanges object with experiment insertions
+#' @param background_gr GRanges object with background insertions
+#' @param regions_gr GRanges object with regions to quantify
+#' @param deduplicate_experiment Whether to deduplicate experiment insertions (default: TRUE)
+#' @param pseudocount Pseudocount for calculations (default: 0.1)
+#' @return GRanges object with regions and statistics as metadata columns
+enrichment_analysis <- function(experiment_gr,
+                                background_gr,
+                                regions_gr,
+                                deduplicate_experiment = TRUE,
+                                pseudocount = 0.1) {
+
+  message("Starting enrichment analysis...")
+
+  # Validate inputs
+  if (!inherits(experiment_gr, "GRanges")) {
+    stop("experiment_gr must be a GRanges object")
+  }
+  if (!inherits(background_gr, "GRanges")) {
+    stop("background_gr must be a GRanges object")
+  }
+  if (!inherits(regions_gr, "GRanges")) {
+    stop("regions_gr must be a GRanges object")
+  }
+
+  # Count overlaps for experiment (with deduplication if requested)
+  message("Counting experiment overlaps...")
+  if (deduplicate_experiment) {
+    message("   Deduplication: ON")
+  } else {
+    message("   Deduplication: OFF")
+  }
+
+  experiment_counts <- count_overlaps(
+    experiment_gr, regions_gr,
+    deduplicate = deduplicate_experiment
+  )
+
+  # Count overlaps for background (never deduplicated)
+  message("Counting background overlaps...")
+  message("   Deduplication: OFF (background should not be deduplicated)")
+
+  background_counts <- count_overlaps(
+    background_gr, regions_gr,
+    deduplicate = FALSE
+  )
+
+  # Calculate total hops AFTER any deduplication
+  if (deduplicate_experiment) {
+    experiment_gr_dedup <- deduplicate_granges(experiment_gr)
+    total_experiment_hops <- length(experiment_gr_dedup)
+  } else {
+    total_experiment_hops <- length(experiment_gr)
+  }
+
+  total_background_hops <- length(background_gr)
+
+  message("Total experiment hops: ", total_experiment_hops)
+  message("Total background hops: ", total_background_hops)
+
+  if (total_experiment_hops == 0) {
+    stop("Experiment data is empty")
+  }
+  if (total_background_hops == 0) {
+    stop("Background data is empty")
+  }
+
+  # Add counts and totals as metadata columns
+  mcols(regions_gr)$experiment_hops <- as.integer(experiment_counts)
+  mcols(regions_gr)$background_hops <- as.integer(background_counts)
+  mcols(regions_gr)$total_experiment_hops <- as.integer(total_experiment_hops)
+  mcols(regions_gr)$total_background_hops <- as.integer(total_background_hops)
+
+  # Calculate statistics
+  message("Calculating enrichment scores...")
+  mcols(regions_gr)$callingcards_enrichment <- calculate_enrichment(
+    total_background_hops = total_background_hops,
+    total_experiment_hops = total_experiment_hops,
+    background_hops = background_counts,
+    experiment_hops = experiment_counts,
+    pseudocount = pseudocount
+  )
+
+  message("Calculating Poisson p-values...")
+  mcols(regions_gr)$poisson_pval <- calculate_poisson_pval(
+    total_background_hops = total_background_hops,
+    total_experiment_hops = total_experiment_hops,
+    background_hops = background_counts,
+    experiment_hops = experiment_counts,
+    pseudocount = pseudocount
+  )
+
+  message("Calculating log Poisson p-values...")
+  mcols(regions_gr)$log_poisson_pval <- calculate_poisson_pval(
+    total_background_hops = total_background_hops,
+    total_experiment_hops = total_experiment_hops,
+    background_hops = background_counts,
+    experiment_hops = experiment_counts,
+    pseudocount = pseudocount,
+    log.p = TRUE
+  )
+
+  message("Calculating hypergeometric p-values...")
+  mcols(regions_gr)$hypergeometric_pval <- calculate_hypergeom_pval(
+    total_background_hops = total_background_hops,
+    total_experiment_hops = total_experiment_hops,
+    background_hops = background_counts,
+    experiment_hops = experiment_counts
+  )
+
+  # Calculate adjusted p-values
+  message("Calculating adjusted p-values...")
+  mcols(regions_gr)$poisson_qval <- p.adjust(mcols(regions_gr)$poisson_pval, method = "fdr")
+  mcols(regions_gr)$hypergeometric_qval <- p.adjust(mcols(regions_gr)$hypergeometric_pval, method = "fdr")
+
+  message("Analysis complete!")
+
+  return(regions_gr)
+}
+
+
+# Example Usage -----------------------------------------------------------
+
+# This is a template for how to use these functions
+# Uncomment and modify for your actual data
+
+# # Load your data (BED3+ format: chr, start, end, ...)
+experiment_gr = arrow::read_parquet("~/code/hf/callingcards/genome_map/batch=run_5801/part-0.parquet") %>%
+  filter(id == 707) %>%
+  dplyr::rename(score = depth) %>%
+  relocate(chr, start, end, id, score, strand) %>%
+  mutate(depth = scales::rescale(score, to = c(1,1000))) %>%
+  bed_to_granges()
+
+background_gr <- read_tsv("~/code/hf/callingcards/adh1_background_ucsc.qbed") %>%
+  mutate(id = "adh1_bg",
+         score = scales::rescale(depth, to = c(1,1000))) %>%
+  dplyr::select(chr, start, end, id, score, strand) %>%
+  bed_to_granges()
+
+regions_gr <- read_tsv("~/code/hf/yeast_genome_resources/yiming_promoters.bed",
+                        col_names = c('chr', 'start', 'end', 'locus_tag', 'score', 'strand'))  %>%
+  bed_to_granges()
+
+# # Run analysis with deduplication (default for calling cards)
+results <- enrichment_analysis(
+  experiment_gr = experiment_gr,
+  background_gr = background_gr,
+  regions_gr = regions_gr,
+  deduplicate_experiment = TRUE,
+  pseudocount = 0.1
+)
+
+
+# id 9 corresponds to the binding sample -- can get from genome_map and
+# annotated_feature metadata
+#
+# NOTE: there are some expected differences due to a change in how I am handling
+# the promoter boundaries. The implementation here is correct -- please use
+# this from now on. If you need to compare or doubt something, please let
+# me konw
+#
+# curr_db_annotated_feature = arrow::read_parquet("~/code/hf/callingcards/annotated_features/batch=run_5801/part-0.parquet") %>%
+#   filter(id == 9)
+#
+# comp_df = curr_db_annotated_feature %>%
+#   select(target_locus_tag, experiment_hops,
+#          background_hops, background_total_hops,
+#          experiment_total_hops) %>%
+#   left_join(results %>%
+#               as_tibble() %>%
+#               select(locus_tag, total_background_hops,
+#                      total_experiment_hops,
+#                      experiment_hops, background_hops) %>%
+#               dplyr::rename(target_locus_tag = locus_tag,
+#                             new_exp_hops = experiment_hops,
+#                             new_bg_hops = background_hops,
+#                             new_bg_total = total_background_hops,
+#                             new_expr_total = total_experiment_hops))