gptomics/bioskills/enhancer-gene-linking

Version Compatibility

Reference examples tested with: ABC-Enhancer-Gene-Prediction 0.2.2+ (Engreitz lab), ENCODE-rE2G v1.0+ (EngreitzLab), Cicero 1.20+, GenomicInteractions 1.36+, FitHiChIP 9.1+, HiC-Pro 3.1+, FAN-C 0.9+, MACS3 3.0+, samtools 1.19+, bedtools 2.31+.

Verify before use:

• CLI: <tool> --version then <tool> --help to confirm flags
• R: packageVersion('<pkg>') then ?function_name to verify parameters
• Python: pip show <package> then help(module.function) to check signatures

If code throws unexpected errors, introspect the installed package and adapt rather than retrying.

Enhancer-Gene Linking

"Which gene does this distal accessible region regulate?" -> Predict the enhancer's target gene using a model that combines accessibility activity, 3D contact frequency, and (optionally) sequence-based chromatin predictions. Output is a per-(enhancer, gene) score that can be thresholded for high-confidence calls.

• CLI: ABC pipeline (run.neighborhoods.py, predict.py from Engreitz lab)
• CLI: ENCODE-rE2G (Snakemake-based; ENCODE 4 enhancer-gene standard)
• R: Cicero (ATAC-only; covered in atac-seq/co-accessibility)
• CLI: FitHiChIP / hichipper for HiChIP H3K27ac loops
• Database: EpiMap (Boix 2021), GeneHancer, FANTOM5 (pre-computed reference)

ABC and ENCODE-rE2G are the canonical predictors when Hi-C/Micro-C data is available. Cicero is the ATAC-only fallback. CRISPRi-FlowFISH (Fulco 2019) is the gold-standard experimental validation.

Algorithmic Taxonomy

Methodology evolves; verify against current Engreitz lab releases (ABC), ENCODE 4 publications (ENCODE-rE2G), and Mumbach 2017 (HiChIP) before locking pipelines.

ABC Mathematics

For each candidate (enhancer E, gene G) pair within the cis window (default 5 Mb):

ABC(E -> G) = Activity_E * Contact_E,G / sum_{all e in window}(Activity_e * Contact_e,G)

• Activity_E = ATAC reads at E * H3K27ac reads at E (geometric mean of normalized signals; reflects "enhancer strength")
• Contact_E,G = Hi-C/Micro-C contact frequency from E to G's TSS (after distance-correction)
• Window = +/- 5 Mb cis (default; ENCODE-rE2G uses 1 Mb)

Threshold typical: ABC >= 0.02 for high-confidence; >= 0.01 for exploratory.

When Hi-C is unavailable, ABC uses an "average contact" averaged across 10 ENCODE Hi-C cell types as proxy (Nasser 2021); it performs comparably to cell-type-matched Hi-C. The alternative powerlaw approximation of contact-vs-distance is the Fulco 2019 fallback.

ENCODE-rE2G Differences from ABC

ENCODE-rE2G (Gschwind et al 2023, bioRxiv) is a reformulation:

• Logistic regression trained on CRISPRi-FlowFISH ground truth (~10 cell types)
• Features: ABC score components + 3D contact + distance + activity ratios
• Multiple feature configurations: "abc-features", "no-hic-features" for cells without 3D data
• Output: Per-pair probability of regulatory connection
• Pre-trained models for ENCODE cell lines; logistic params vary by cell type

ENCODE-rE2G generally outperforms ABC at CRISPRi recall, especially at modest distances (50-500 kb). For ENCODE cell types, prefer ENCODE-rE2G; for novel cell types, ABC remains the default.

Per-Tool Failure Modes

ABC -- Wrong cell-type-matched Hi-C

Trigger: Using K562 Hi-C contact when actual cell type is GM12878.

Mechanism: Contact frequencies differ across cell types at compartment and TAD boundaries; using mismatched Hi-C produces wrong ABC scores.

Symptom: ABC predictions concentrate at known K562-specific loci even when ATAC data is from GM12878.

Fix: Use cell-type-matched Hi-C or Micro-C. If unavailable, ABC's "average HiC" (10-cell-type pooled) is the documented fallback with acknowledged degradation. Document the proxy in methods.

ABC -- H3K27ac normalization

Trigger: H3K27ac ChIP-seq with different sequencing depth than ATAC.

Mechanism: ABC's "Activity" is the geometric mean of accessibility and H3K27ac signals; both must be normalized to the same scale.

Symptom: Activity scores skewed; some peaks have very high activity from H3K27ac alone, others from ATAC alone.

Fix: Normalize both signals to reads-per-million in peaks (RPM-IP) before combining. Use ABC's --qnorm flag with a quantile-normalization reference file (e.g. --qnorm reference/EnhancersQNormRef.K562.txt from the ABC repo).

ENCODE-rE2G -- Cell type not in pre-trained set

Trigger: Running pre-trained model on a primary cell type not in CRISPRi training.

Mechanism: Logistic regression coefficients learned from ENCODE cell types may not transfer to primary tissues.

Fix: Use the closest ENCODE cell type (myeloid lineage -> K562; lymphoid -> GM12878; hepatic -> HepG2). Document the proxy. For high-stakes use, custom retraining requires CRISPRi-FlowFISH data.

Cicero -- No Hi-C concordance benchmark

Trigger: Reporting Cicero connections as enhancer-gene calls without external validation.

Mechanism: Cicero is statistical co-accessibility; correlation with Hi-C 3D contacts is ~30-50%. Many strong Cicero connections are NOT Hi-C-validated.

Fix: When Hi-C is available, cross-validate; report both. When only ATAC, use Cicero with the explicit caveat that connections are co-accessibility hypotheses, not contact predictions.

HiChIP -- Loop calling threshold

Trigger: Default FitHiChIP at FDR < 0.05.

Mechanism: HiChIP loops are abundant (10k-100k per dataset); FDR alone produces a long tail of weak loops.

Fix: Threshold at FDR < 0.05 AND number of contacts per loop >= 5; or use the top N most significant where N = expected number of loops based on cell type.

EpiMap / GeneHancer -- Cell-type-agnostic limitation

Trigger: Using EpiMap or GeneHancer pre-computed pairs for a specific cell type.

Mechanism: These references aggregate across many tissues / experiments; cell-type-specific connections are diluted.

Fix: Use as a baseline / sanity check, not as the primary call. ABC or ENCODE-rE2G in the actual cell type is preferred.

Decision Tree by Available Data

ABC Standard Pipeline

Goal: Compute per-(enhancer, gene) ABC scores combining ATAC accessibility, H3K27ac activity, and Hi-C contact.

Approach: Define non-promoter candidate enhancers from ATAC peaks, run ABC neighborhoods (which counts reads directly from the ATAC/H3K27ac BAMs) to compute per-candidate activity, then run ABC predict against a Hi-C contact matrix and threshold the per-pair ABC score.

# 1. (Optional, browser tracks only) ATAC/H3K27ac bigWigs -- ABC neighborhoods below reads the BAMs directly, not bigWigs
bamCoverage --bam atac.bam --outFileName atac.bw --binSize 50 --normalizeUsing RPGC \
    --effectiveGenomeSize 2701495711 --numberOfProcessors 8
 
# 2. Define enhancer candidates (typically MACS narrowPeak from ATAC)
# Filter to non-promoter regions
bedtools intersect -v -a atac_peaks.narrowPeak -b promoter_regions.bed > candidate_enhancers.bed
 
# 3. Run ABC neighborhoods (compute Activity per candidate)
# Script path: legacy ABC = src/run.neighborhoods.py; Snakemake-based modern = workflow/scripts/run.neighborhoods.py
python /path/ABC-Enhancer-Gene-Prediction/workflow/scripts/run.neighborhoods.py \
    --candidate_enhancer_regions candidate_enhancers.bed \
    --genes refseq_protein_coding.bed \
    --H3K27ac h3k27ac.bam \
    --DHS atac.bam \
    --chrom_sizes hg38.chrom.sizes \
    --chrom_sizes_bed hg38.chrom.sizes.bed \
    --ubiquitously_expressed_genes Genes.ubiquitously_expressed.txt \
    --cellType MyCellType \
    --outdir abc_out/
 
# 4. Run ABC predictions (Activity * Contact) -- generates ALL unthresholded links
python /path/ABC-Enhancer-Gene-Prediction/workflow/scripts/predict.py \
    --enhancers abc_out/EnhancerList.txt \
    --genes abc_out/GeneList.txt \
    --hic_file hic_data/ \
    --hic_type avg \
    `# --hic_type choices: hic | juicebox | bedpe | avg -- must match the Hi-C input format` \
    --hic_resolution 5000 \
    --hic_pseudocount_distance 5000 \
    `# --hic_pseudocount_distance (required): powerlaw fit at this distance is added as a pseudocount (config default 5000)` \
    --chrom_sizes hg38.chrom.sizes \
    --score_column ABC.Score \
    --cellType MyCellType \
    --outdir abc_out/Predictions/
 
# predict.py writes EnhancerPredictionsAllPutative.tsv.gz (all unthresholded E-G links).
# 5. Threshold at ABC.Score >= 0.02. The ABC Snakemake pipeline runs filter_predictions.py with its
# full set of --output_* arguments; for a standalone cut, select by the ABC.Score column (by header):
zcat abc_out/Predictions/EnhancerPredictionsAllPutative.tsv.gz | \
    awk -F'\t' 'NR==1{for(i=1;i<=NF;i++)if($i=="ABC.Score")c=i; print; next} $c>=0.02' \
    > abc_out/Predictions/EnhancerPredictions_thresholded.tsv

ABC.Score >= 0.02 is the standard threshold validated in Fulco 2019 against CRISPRi-FlowFISH; >= 0.04 is a stricter cut sometimes used in the ABC pipeline documentation for higher precision (no separate primary-paper calibration).

ENCODE-rE2G