Chip Seq

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

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

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