Version Compatibility

Reference examples tested with: ChIPseeker 1.38+, GenomicFeatures 1.54+, rtracklayer 1.62+, HOMER 4.11+, rGREAT 2.4+, chipenrich 2.26+, pyranges 0.0.129+, pandas 2.2+.

ENCODE cCRE registry expanded to 2.37M human and 967k mouse elements (Moore JE et al 2026 Nature). SCREEN web app at screen.encodeproject.org provides browser access; ENCODE provides bed files for batch annotation.

Peak Annotation

"What genes and regulatory elements do my peaks correspond to?" -> Assign each peak to a genomic feature (promoter, exon, intron, intergenic), its target gene (via nearest-TSS or host-gene), and where applicable an ENCODE cCRE class (PLS/pELS/dELS/CA-CTCF/CA-H3K4me3).

• R (gene-feature): ChIPseeker::annotatePeak(peaks, TxDb=txdb)
• CLI (gene-feature): annotatePeaks.pl peaks.bed hg38 -gtf annotation.gtf
• Python (custom): pyranges + pandas
• R (cCRE classification): intersect peaks with ENCODE cCRE BED from SCREEN
• R (gene-set enrichment): rGREAT::great() or chipenrich::chipenrich()

The single biggest source of misinterpretation is the nearest-TSS vs host-gene distinction (see below). For enhancer-driven biology, ENCODE-rE2G or ABC (in atac-seq/enhancer-gene-linking) is more accurate than nearest-TSS.

Choosing an Annotation Approach

| Context | Recommended | Why | |---------|-------------|-----| | Standard genome, pre-built annotations available | ChIPseeker with TxDb package | Simplest; automatic gene symbol mapping via annoDb | | Custom or project-specific GTF | ChIPseeker + makeTxDbFromGFF, HOMER -gtf, or pyranges | All three handle custom annotations | | HOMER already in pipeline | HOMER annotatePeaks.pl | Reuses tag directory; combined with motif workflow | | Fine-grained control | pyranges (Python) | Full control over priority rules, distance calculation | | Enhancer peaks (distal regulatory) | GREAT / rGREAT | Regulatory domain assignment (basal + extension), not just nearest | | Cell-type-specific enhancer-gene linking | ENCODE-rE2G | Modern (2024); ABC-trained logistic regression with chromatin context | | Gene-set enrichment with locus-length adjustment | chipenrich / Broad-Enrich | Corrects for systematic gene-length bias in peak assignment | | Compare against ENCODE cCRE atlas | SCREEN cCRE BED intersect | Cross-reference standard regulatory registry | | Promoter-coverage decomposition | bedtools intersect with TSS windows | Quick stats per peak set |

Critical: Use the same annotation source as the alignment (UCSC knownGene TxDb with GENCODE GTF alignment causes mismatches). When a specific GTF is provided, use it directly via makeTxDbFromGFF rather than a mismatched pre-built TxDb package.

Nearest-TSS vs Host-Gene Convention

Peak annotation involves two decisions that should be coupled but often aren't:

1. Which gene to assign (target gene)
2. What feature the peak overlaps (promoter / exon / intron / intergenic)

Default tools decouple these, producing internally inconsistent annotations.

| Convention | Gene from | Feature from | Tools | |------------|-----------|---------------|-------| | Nearest-TSS (default) | Gene with closest TSS | Physical overlap at peak center | ChIPseeker overlap='TSS' (default), HOMER | | Host-gene priority | Gene whose body contains the peak | Same gene's features | ChIPseeker overlap='all' |

Example failure: Peak inside gene A's intron, near gene B's TSS. Default tools report nearest_gene=B, feature=intron — but the intron belongs to gene A, not gene B. The annotation is internally inconsistent.

Choosing per Biology

| Context | Convention | Rationale | |---------|-----------|-----------| | Distal TF binding (enhancers) | Nearest-TSS, but prefer ENCODE-rE2G / ABC | Enhancers can regulate gene A despite sitting in gene B's intron | | Histone marks in gene bodies (H3K36me3, H3K27me3) | Host-gene | Mark reflects host transcriptional state | | Promoter-associated marks (H3K4me3, H3K27ac at promoters) | Either | Most peaks at promoters where conventions agree | | Custom annotation against project GTF | Host-gene | Internal consistency | | Reproducing published HOMER results | Nearest-TSS | Matches HOMER default |

When a task says "nearest gene," clarify which definition. For most annotation purposes where gene + feature should be consistent, use host-gene; for distal enhancer biology, use a proper enhancer-gene linker (ENCODE-rE2G, ABC).

Coordinate Systems and TSS

BED uses 0-based half-open [start, end). GTF uses 1-based closed [start, end]. Mixing without conversion shifts annotations by one base.

Peak center (BED): (start + end) // 2

TSS from GTF (1-based to 0-based):

• Plus-strand: tss_0based = start - 1
• Minus-strand: tss_0based = end

Signed distance (negative = upstream of TSS):

• Plus-strand: distance = peak_center - tss
• Minus-strand: distance = -(peak_center - tss)

ChIPseeker (R)

Goal: Assign each ChIP-seq peak to a gene and a feature category using a transcript database.

Approach: Load the TxDb (pre-built or custom-built from GTF), pass peaks to annotatePeak() with the desired tssRegion window and overlap convention (host-gene vs nearest-TSS), then export the annotated data frame with gene symbols mapped from annoDb or the original GTF.

Standard genome:

library(ChIPseeker)
library(TxDb.Hsapiens.UCSC.hg38.knownGene)
library(org.Hs.eg.db)

peaks <- readPeakFile('peaks.narrowPeak')
peak_anno <- annotatePeak(peaks,
                           TxDb = TxDb.Hsapiens.UCSC.hg38.knownGene,
                           tssRegion = c(-2000, 2000),
                           annoDb = 'org.Hs.eg.db',
                           overlap = 'all')   # host-gene convention
anno_df <- as.data.frame(peak_anno)

Custom GTF (use makeTxDbFromGFF; map symbols from original GTF since custom TxDb objects lack annoDb mappings):

library(GenomicFeatures)
library(rtracklayer)

txdb <- makeTxDbFromGFF('genes.gtf.gz', format = 'gtf')
peaks <- readPeakFile('peaks.bed')
peak_anno <- annotatePeak(peaks, TxDb = txdb, tssRegion = c(-2000, 2000),
                           overlap = 'all')

gtf <- import('genes.gtf.gz')
gene_map <- unique(data.frame(
    gene_id = sub('\\..*', '', gtf$gene_id),
    symbol = gtf$gene_name, stringsAsFactors = FALSE))
gene_map <- gene_map[!is.na(gene_map$symbol), ]
anno_df <- as.data.frame(peak_anno)
anno_df$gene_id_base <- sub('\\..*', '', anno_df$geneId)
anno_df$SYMBOL <- gene_map$symbol[match(anno_df$gene_id_base, gene_map$gene_id)]

GENCODE gene IDs have version suffixes (ENSG00000142192.25); strip before joining.

Promoter window: tssRegion = c(-2000, 2000) is common; c(-3000, 3000) is ChIPseeker default. Match to analysis requirements.

Feature priority: Default Promoter > 5'UTR > 3'UTR > Exon > Intron > Downstream > Intergenic. A peak in both a promoter (gene A) and an intron (gene B) receives "Promoter (gene A)" by default.

HOMER annotatePeaks.pl (CLI)

# Standard genome (HOMER's installed annotation)
annotatePeaks.pl peaks.bed hg38 > annotated.txt

# Custom GTF (overrides HOMER's default)
annotatePeaks.pl peaks.bed hg38 -gtf genes.gtf > annotated.txt

# Without installed genome, GTF only
annotatePeaks.pl peaks.bed none -gtf genes.gtf > annotated.txt

# Generate annotation statistics
annotatePeaks.pl peaks.bed hg38 -gtf genes.gtf -annStats stats.txt > annotated.txt

HOMER's 19-column output: columns 8 (Annotation), 10 (Distance to TSS), 16 (Gene Name) are the primary annotation columns.

HOMER promoter window is fixed at -1kb / +100bp — not configurable via flags. For custom windows, reclassify using the Distance to TSS column post-hoc.

ENCODE cCRE Classification

The ENCODE Registry of candidate cis-Regulatory Elements (cCREs) provides 2.37M human + 967k mouse elements. Registry V4 uses an 8-class scheme (the older V3 "CTCF-only" and "DNase-H3K4me3" were renamed CA-CTCF and CA-H3K4me3):

| Class | Definition | Marker pattern | |-------|------------|-----------------| | PLS (Promoter-Like Signature) | ≤ 200 bp of annotated TSS; high DNase + high H3K4me3 | DNase + H3K4me3 | | pELS (Proximal Enhancer-Like Signature) | ≤ 2 kb of TSS; enhancer-like (DNase + H3K27ac, low H3K4me3) | DNase + H3K27ac | | dELS (Distal Enhancer-Like Signature) | > 2 kb of TSS; enhancer-like | DNase + H3K27ac | | CA-H3K4me3 | Chromatin-accessible + H3K4me3, not TSS-proximal | DNase + H3K4me3 | | CA-CTCF | Chromatin-accessible + CTCF (potential boundary) | DNase + CTCF | | CA-TF | Chromatin-accessible + TF binding | DNase + TF | | CA | Chromatin-accessible only | DNase | | TF | TF-bound, not highly accessible | TF |

# Download ENCODE cCRE BED from SCREEN (GRCh38, expanded Registry-V4, uncompressed)
wget https://downloads.wenglab.org/Registry-V4/GRCh38-cCREs.bed

# Intersect peaks with cCRE; -wa preserves peak coords, -wb adds cCRE class
bedtools intersect -a peaks.narrowPeak -b GRCh38-cCREs.bed -wa -wb \
    > peaks_ccre.tsv

Cross-referencing peaks against cCREs:

• Indicates whether peaks overlap canonical regulatory elements
• Provides the cCRE class (PLS / pELS / dELS / CA-CTCF / CA-H3K4me3)
• Cell-type-specific activity profiles available via SCREEN web app

GREAT / rGREAT (Regulatory Domain Gene-Set Enrichment)

GREAT (McLean 2010) addresses two problems with standard gene-set enrichment on peaks:

1. Peak-to-gene assignment via regulatory domains (not nearest TSS)
2. Statistical correction for region-locus length bias

Regulatory domain rules (default):

• Basal domain: -5 kb / +1 kb of TSS
• Extension: up to 1 Mb in each direction, OR until reaching neighbor's basal domain
• Each peak is assigned to ALL genes whose regulatory domain it overlaps (not just nearest)

library(rGREAT)

# Submit peaks for regulatory-domain gene-set enrichment
res <- great(gr = peaks, gene_sets = 'GO:BP', tss_source = 'TxDb.Hsapiens.UCSC.hg38.knownGene',
              biomart_dataset = 'hsapiens_gene_ensembl')

# Top enriched gene sets
table_results <- getEnrichmentTable(res)
head(table_results)

# Visualization (local great() returns a GreatObject -> plotRegionGeneAssociations)
plotVolcano(res)
plotRegionGeneAssociations(res)

GREAT is most appropriate for distal regulatory elements (enhancer ChIP, ATAC). For promoter-focused marks (H3K4me3), ChIP-Enrich is more standard.

ChIP-Enrich (Locus-Length-Adjusted Gene-Set Enrichment)

Welch 2014: standard gene-set enrichment on peak-associated genes systematically over-counts long genes. ChIP-Enrich models locus length as a covariate.

library(chipenrich)

res <- chipenrich(peaks = 'peaks.bed', genome = 'hg38',
                   genesets = 'GOBP', locusdef = 'nearest_tss',
                   out_name = 'chipenrich_out', n_cores = 4)
# Locus definitions: nearest_tss, nearest_gene, exon, intron, 1kb, 5kb, 10kb
# method= accepts chipenrich (default) or fet; broadenrich() and polyenrich() are separate functions

For broad marks (H3K27me3, H3K9me3): use the separate broadenrich(peaks = 'peaks.bed', genome = 'hg38', genesets = 'GOBP', locusdef = 'nearest_tss') function, which accounts for region width.

ENCODE-rE2G (Modern Enhancer-Gene Linking)

ENCODE-rE2G (2024) replaces ABC for cell types with ENCODE data. Cell-type-specific logistic-regression weights map distal enhancer peaks to target genes with higher accuracy than nearest-TSS or basal+extension.

See atac-seq/enhancer-gene-linking for full workflow; the same model applies to ChIP-seq enhancer marks (H3K27ac, H3K4me1, H3K4me2).

Per-Tool Failure Modes

ChIPseeker -- TxDb / annoDb genome mismatch

Trigger: Using hg19 TxDb on hg38-aligned BAMs / peaks.

Mechanism: Silent; ChIPseeker doesn't verify genome assembly.

Symptom: Annotated gene symbols look reasonable but distance-to-TSS is wrong; promoter / intron classifications drift.

Fix: Match TxDb to BAM alignment genome explicitly; verify with seqlevels(peaks) == seqlevels(txdb).

ChIPseeker -- Default overlap='TSS' decouples gene from feature

Trigger: Default annotation call on peaks in gene bodies.

Mechanism: overlap='TSS' assigns nearest gene by TSS; feature classification is independent of that gene.

Symptom: Annotation reports nearest_gene=X, feature=intron where the intron belongs to a different gene.

Fix: Pass overlap='all' for host-gene-consistent annotation; or accept TSS-only convention and clarify in methods.

ChIPseeker -- Custom TxDb has no annoDb

Trigger: Building TxDb from GTF and passing annoDb='org.Hs.eg.db'.

Mechanism: Custom TxDb lacks the gene_id-to-symbol mapping that org.Hs.eg.db provides; ChIPseeker silently returns NA for symbols.

Fix: Map symbols separately from the original GTF after annotation; strip Ensembl version suffixes before joining.

HOMER -- Hard-coded promoter window

Trigger: Needing a 2 kb or 5 kb promoter window with HOMER.

Mechanism: HOMER's promoter classification is hard-coded to -1 kb / +100 bp; not configurable.

Fix: Post-hoc reclassify using Distance to TSS column:

awk -F'\t' 'NR>1 { dist = ($10 < 0) ? -$10 : $10; \
    feat = (dist <= 2000) ? "promoter_custom" : $8; \
    print $2, $3, $4, $16, $10, feat }' OFS='\t' annotated.txt

GREAT -- Default regulatory domain inappropriate for some species / cell types

Trigger: Using default basal+extension on insect or compact-genome data.

Mechanism: 1 Mb maximum extension assumes vertebrate-scale enhancer-target distances; not appropriate for organisms with shorter regulatory ranges.

Fix: Adjust extension parameter; for non-default species, configure regulatory domain explicitly.

GREAT -- Hyper-ChIPable peaks inflate enrichment

Trigger: Including unfiltered peaks at rRNA / housekeeping / mtDNA in GREAT analysis.

Mechanism: Hyper-ChIPable artifacts are enriched at highly-transcribed loci; GREAT assigns them to associated genes, inflating GO terms for "translation" and "ribosomal" categories.

Symptom: Top enriched GO terms always include "ribosomal", "translation", "mitochondrion" regardless of biology.

Fix: Blacklist filter + custom hyper-ChIPable filter (top-1% input signal) before GREAT.

ENCODE cCRE -- Cell-type-agnostic vs specific

Trigger: Using the master cCRE BED (cell-type-agnostic) to claim cell-type-specific regulatory activity.

Mechanism: Master cCRE BED is the union across all cell types. Specific activity profile per cell type is a separate dataset.

Fix: Use SCREEN web app or per-cell-type activity profiles for cell-type-specific claims.

Reconciliation: When Methods Disagree

| Pattern | Likely cause | Action | |---------|--------------|--------| | ChIPseeker nearest-TSS gene ≠ HOMER nearest gene | Different TSS reference; HOMER uses RefSeq | Verify both use same TxDb / RefSeq + UCSC knownGene | | GREAT enrichment ≠ ChIP-Enrich enrichment | GREAT uses regulatory domain; ChIP-Enrich uses locus length adjustment | Both are valid; use GREAT for distal regulatory, ChIP-Enrich for promoter-focused | | Peak overlaps cCRE but classified differently than expected | Cell-type-specific activity profile not used | Check SCREEN per-cell-type profile | | Enhancer peak's nearest gene differs from ENCODE-rE2G target | ENCODE-rE2G uses cell-type chromatin context | Use ENCODE-rE2G for cell-type-specific enhancer-gene claims |

Common Errors

| Error / symptom | Cause | Solution | |-----------------|-------|----------| | seqlevels mismatch in ChIPseeker | chr vs no-chr naming | seqlevelsStyle(peaks) <- 'UCSC' | | Gene symbols all NA in ChIPseeker | Custom TxDb without annoDb | Map symbols from original GTF | | HOMER reports "no annotation" | Genome not installed | perl configureHomer.pl -install hg38 | | rGREAT timeout | Large peak set + slow biomart | Use pre-computed gene sets; lower peak count | | chipenrich slow | Default locusdef computed on-the-fly | Use built-in locusdef shortcuts (nearest_tss, 1kb) | | pyranges feature-overlap result missing strand | pyranges 0.x conversion drops strand by default | Pass strandedness='same' to overlap operations |

References

• Yu G et al 2015 Bioinformatics 31:2382 (ChIPseeker)
• Heinz S et al 2010 Mol Cell 38:576 (HOMER annotatePeaks)
• McLean CY et al 2010 Nat Biotechnol 28:495 (GREAT)
• Gu Z 2023 Bioinformatics 39:btac745 (rGREAT)
• Welch RP et al 2014 Nucleic Acids Res 42:e105 (ChIP-Enrich)
• Cavalcante RG, Lee C, Welch RP, ... Sartor MA 2014 Bioinformatics 30:i393-i400 (Broad-Enrich)
• ENCODE Project Consortium 2020 Nature 583:699 (cCRE registry v1)
• Moore JE et al 2026 Nature (expanded cCRE registry, 2.37M human + 967k mouse elements)
• Fulco CP et al 2019 Nat Genet 51:1664 (ABC model precursor to ENCODE-rE2G)
• Kundaje lab / ENCODE 2024 (ENCODE-rE2G)
• SCREEN: screen.encodeproject.org

Related Skills

• chip-seq/peak-calling - Generate peaks for annotation
• chip-seq/chipseq-qc - Filter hyper-ChIPable peaks before GREAT / chipenrich
• chip-seq/super-enhancers - Annotate SE-associated genes
• chip-seq/differential-binding - Annotate differential peaks
• atac-seq/enhancer-gene-linking - ENCODE-rE2G workflow for cell-type-specific E-G linking
• pathway-analysis/go-enrichment - Standard GO enrichment on peak-associated genes
• pathway-analysis/reactome-pathways - Reactome pathway enrichment
• genome-intervals/gtf-gff-handling - Parse and convert GTF/GFF
• genome-intervals/proximity-operations - bedtools closest and window operations