Peak Calling

What the source does

• FilesystemNone observedNo reads or writes in the source
• NetworkNone observedNo outbound calls in the source
• ShellNone observedNo commands run in the source
• DependenciesPinnedNothing floats
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Five audit layers

Gates applied: no behavioural pass.

Audit 0.4.1 · source sha c8c1fc3a2b13 · 19 Oct 2026Report an issue or request a re-scan

• PublisherGPTomics GitHub organizationOwner of the repository this skill is read from
• Source folderGPTomics/bioSkills/chip-seq/peak-callingThe exact folder we audited
• LicenceMITAs the repository states it
• Commit pinned for this verdictc8c1fc3a2b13A new audit runs when the source changes
• Repository statusArchivedLast push 65 days ago
• Sandboxed runNot runSkills are read, not executed; layer L4 is skipped

Its description: Calls ChIP-seq peaks with MACS3, MACS2, HOMER, or SPP across narrow (TF) and broad (histone) modes. Handles input control matching, fragment-size modeling vs --nomodel, effective genome size, ENCODE-style IDR vs naive…

---
name: bio-chipseq-peak-calling
description: Calls ChIP-seq peaks with MACS3, MACS2, HOMER, or SPP across narrow (TF) and broad (histone) modes. Handles input control matching, fragment-size modeling vs --nomodel, effective genome size, ENCODE-style IDR vs naive overlap, hyper-ChIPable artifacts, and aligner-specific shifts. Use when calling peaks from ChIP-seq alignments, choosing between narrow vs broad mode for a histone mark, deciding model vs nomodel for low-depth data, applying ENCODE pseudoreplicate IDR, or reconciling MACS vs HOMER vs SPP results.
tool_type: cli
primary_tool: macs3
---

## Version Compatibility

Reference examples tested with: MACS3 3.0.4+, MACS2 2.2.9+, HOMER 4.11+, SPP 1.16+, samtools 1.19+, bedtools 2.31+, IDR 2.0.4+.

Before running, verify versions: `<tool> --version` and `<tool> --help` to confirm flags. If a flag is missing, check the changelog — MACS2->MACS3 is API-compatible for `callpeak` but `predictd`, `bdgpeakcall`, and `hmmratac` differ.

# ChIP-seq Peak Calling

**"Identify protein-DNA binding sites from ChIP-seq alignments"** -> Detect statistically enriched genomic regions by comparing IP signal to input control (or genomic background), with peak shape (narrow/broad) determined by target biology (TF vs histone mark).

- CLI (ENCODE TF default): `macs2 callpeak -t chip.bam -c input.bam -f BAM -g hs -n sample --keep-dup all -p 1e-2`
- CLI (ENCODE histone default): same with `--broad --broad-cutoff 0.1` for H3K27me3, H3K9me3, H3K36me3
- CLI (alternative): `macs3 callpeak ...` (API-identical, active development), HOMER `findPeaks tags/ -style histone -i input_tags/`, SPP via phantompeakqualtools wrapper

ENCODE TF pipeline still uses **SPP for peak ranking + IDR**, with MACS2 producing the signal tracks. Histone pipeline uses **MACS2 + naive overlap** (IDR is too conservative for histone signal dynamic range). MACS3 is the actively maintained successor; MACS2 receives only bug fixes.

## Critical Pre-Call Validation

Before any peak calling, three things must be true or the output is unreliable:

1. **Antibody validated** — KO/KD orthogonal control, peptide-array specificity for histone modifications, or vendor-provided CRISPR-validated lot (Epicypher, CST). "ChIP-grade" marketing is not validation. See chipseq-qc.
2. **Fragment-size distribution is sane** — TF ChIP should show sub-nucleosomal (~50-100 bp) enrichment; histone ChIP should show clean mono- (~150) and di-nucleosomal (~300) peaks. Flat distribution = over-sonication; rescue is impossible. Check via `samtools view -f 0x2 sample.bam | awk '{print $9}' | sort | uniq -c`.
3. **Input control matches** — Sonicated input is biased toward open chromatin; MNase input toward nucleosomes. Input from a different library prep batch or fragmentation method introduces bias that subtraction cannot fix.

## Algorithmic Taxonomy

| Tool | Model | Treats fragments as | Strength | Fails when |
|------|-------|---------------------|----------|------------|
| MACS3/MACS2 callpeak | Dynamic local Poisson (max of genome-wide, 1kb, 5kb, 10kb lambda) + BH-FDR | Single-end shifts; PE fragments via BAMPE | Mature, fast, ENCODE-default, narrow + broad modes, integrated signal tracks | Confounds NFR with broad accessible domains; default narrow mode segments broad enrichment; assumes most genome NOT enriched (breaks for genome-wide marks) |
| SPP (Kharchenko 2008) | Strand cross-correlation peak detection + Poisson fold-enrichment | Single-end with cross-corr-derived shift | ENCODE TF caller; integrated NSC/RSC QC; robust for sharp TF peaks | Underperforms for broad marks; older R codebase; phantompeakqualtools wrapper has R-version compatibility issues |
| HOMER `-style factor` | Fixed-width peaks + three sequential filters (control / local / clonal) | Tag positions; auto-estimated width | Fast on tag directories; clonal filter `-C` removes PCR-artifact peaks | Less calibrated p-values; fixed width clips variable-width factor binding |
| HOMER `-style histone` | Variable-width region stitching (500 bp blocks, 1000 bp gap merging); L=0 (no local enrichment) | Tag positions | Captures variable-width histone enrichment; Omnipeak benchmark (Shpynov & Artyomov 2026): outperforms `-style factor` for histone marks including H3K4me3 | Less sensitive than MACS for very sharp TF binding |
| Genrich `-y` (ChIP mode) | q-value on log-transformed p-value, joint replicate model | Whole fragments (PE intervals) | Joint replicate analysis; chrM exclusion via `-e chrM`; auto blacklist via `-E` | Less peer-reviewed than MACS/SPP; thin literature; control handling less mature |
| MACS3 hmmratac | 3-state HMM on fragment-size signal | Fragment-size classes | Best for ATAC, not ChIP | Wrong tool for ChIP; ChIP fragment-size distribution doesn't drive useful HMM states |
| SEACR (Meers 2019) | Empirical threshold on signal block totals | Bedgraph signal blocks | Designed for sparse CUT&RUN/CUT&Tag data; "stringent" mode with IgG strongly preferred | Not for traditional ChIP-seq (assumes near-zero background); see cut-and-run-tag |
| LanceOtron (Hentges 2022) | CNN trained on ENCODE peaks | bigWig signal | Competitive for both narrow and broad without parameter tuning | Newer; less validated; web-only or pip install |

For CUT&RUN / CUT&Tag specifically, see chip-seq/cut-and-run-tag — protocol differences (lower depth, IgG-only control, E. coli spike-in carryover) drive different caller choice (MACS2 + SEACR consensus, not MACS3 alone).

## Decision: Narrow vs Broad

Driven by target biology, not preference. Calling broad mode does not make a sharp signal broad; it changes how MACS stitches adjacent enrichment.

| Target | Mode | Why |
|--------|------|-----|
| Transcription factors (CTCF, p53, GATA1, FOXA1) | Narrow (default) | Discrete motif binding produces sharp peaks |
| H3K4me3, H3K27ac at promoters/enhancers | Narrow | Localized at regulatory elements |
| H3K4me1 at enhancers | Narrow or broad-cutoff 0.1 | Variable; check publis

Excerpt, first 6,000 characters of SKILL.md, verbatim from the audited commit. It is text from someone else’s repository, quoted so you can judge it before your agent reads it.

git clone https://github.com/GPTomics/bioSkills
git -C bioSkills checkout c8c1fc3a2b13

Works in

1. 2026-10-19
   ✓SAFE B 89 / 100

   first audit · source c8c1fc3a2b13

Questions about Peak Calling

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