chip-seq

Research

ChIP-seq peak calling and downstream interpretation with MACS3, signal track export, annotation, motif analysis, and differential binding review.

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Source SKILL.md: https://github.com/Runchuan-BU/BioClaw/blob/HEAD/container/skills/chip-seq/SKILL.md

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ChIP Seq

Version Compatibility

Reference examples assume:

macs3 3.0+
samtools 1.18+
deepTools 3.5+

Before using commands, verify the installed environment:

CLI: macs3 --version, samtools --version, bamCoverage --version
If flags differ, inspect --help and adapt rather than forcing the example unchanged.

Overview

Use this skill for:

narrow or broad peak calling
input-normalized signal tracks
peak annotation
motif follow-up
differential binding review when replicates exist

When To Use This Skill

the user has aligned ChIP and optional input BAM files
the deliverable includes peaks, browser tracks, or motif results
the assay is TF ChIP or histone-mark ChIP and needs standard peak-centric processing

Quick Route

TF or narrow marks: use narrow peak mode first.
H3K27me3, H3K36me3, or other broad marks: use --broad.
Paired-end BAM: prefer -f BAMPE.
No input control: still possible, but report the limitation explicitly.

Progressive Disclosure

Read technical_reference.md for QC gates, narrow-versus-broad logic, and replicate handling.
Read commands_and_thresholds.md for MACS3 commands, parameter defaults, and output file conventions.

Prerequisites

Requirement	Narrow TF-style	Broad histone-style
usable uniquely mapped reads	`>= 10M`	`>= 20M`
matched input recommended	yes	yes
biological replicates recommended	`>= 2`	`>= 2`

Expected Inputs

chip.bam
input.bam when available
reference genome build
chromosome sizes if bigWig export is needed

Expected Outputs

results/peaks/sample_peaks.narrowPeak or .broadPeak
results/peaks/sample_summits.bed
results/tracks/sample_treat_pileup.bw
results/annotation/peak_annotation.tsv
qc/chip_qc_summary.tsv

Starter Pattern

macs3 callpeak \
  -t chip.bam \
  -c input.bam \
  -f BAMPE \
  -g hs \
  -n sample \
  -q 0.01 \
  --outdir results/peaks

Key Parameters

Parameter	Typical value	Meaning
`-f`	`BAM` or `BAMPE`	paired-end should use `BAMPE`
`-g`	`hs`, `mm`, or numeric	effective genome size
`-q`	`0.01` or `0.05`	FDR cutoff for narrow peaks
`--broad`	broad marks only	broad peak mode
`--broad-cutoff`	`0.1`	broad-peak FDR cutoff
`-B --SPMR`	enabled for tracks	bedGraph for normalized signal

Workflow

1. Validate BAMs and replicate structure

Check:

mapped read counts
duplicate burden
whether input control exists
whether the mark is narrow or broad

2. Call peaks with MACS3

narrow marks: -q 0.01 is a good starting point
broad marks: use --broad --broad-cutoff 0.1
paired-end: -f BAMPE

3. Export signal tracks

Use -B --SPMR, sort the resulting bedGraph, then convert to bigWig for browser use.

4. Annotate and inspect peaks

Map peaks to promoters, gene bodies, or distal intervals and review top loci in a genome browser or track plot.

5. Run motif or differential follow-up

Only after peak quality looks credible and replicate structure supports the downstream question.

Output Artifacts

results/
├── peaks/
│   ├── sample_peaks.narrowPeak
│   ├── sample_summits.bed
│   └── sample_model.r
├── tracks/
│   ├── sample_treat_pileup.bdg
│   └── sample_treat_pileup.bw
└── annotation/
    └── peak_annotation.tsv
qc/
└── chip_qc_summary.tsv

Quality Review

TF ChIP-seq FRiP:
- < 0.01 poor
- 0.01-0.05 usable but weak
- > 0.05 generally solid
Histone broad-mark FRiP often differs; compare within assay type rather than against TF expectations.
Use replicate concordance when available. Do not trust a single noisy replicate just because peaks were called.
Check that top peaks occur in plausible loci and not only blacklisted or artifactual regions.

Anti-Patterns

treating broad and narrow marks with the same peak-calling setup
calling peaks on unsorted or low-quality BAMs
presenting motif hits without showing peak quality
hiding that no input control was available

Related Skills

ATAC Seq
Methylation Analysis
Gene Regulatory Networks

Optional Supplements

deeptools
pysam