bio-chipseq-peak-calling

name	bio-chipseq-peak-calling
description	ChIP-seq peak calling using MACS3 (or MACS2). Call narrow peaks for transcription factors or broad peaks for histone modifications. Supports input control, fragment size modeling, and various output formats including narrowPeak and broadPeak BED files.
tool_type	cli
primary_tool	macs3

Peak Calling with MACS3

MACS3 is the actively developed successor to MACS2. Commands are identical except the binary name. MACS2 is in maintenance mode.

Basic Peak Calling

# Call peaks with input control (recommended)
macs3 callpeak -t chip.bam -c input.bam -f BAM -g hs -n sample --outdir peaks/

# MACS2 syntax (identical, for legacy pipelines)
# macs3 callpeak -t chip.bam -c input.bam -f BAM -g hs -n sample --outdir peaks/

Without Input Control

# Not recommended, but possible
macs3 callpeak -t chip.bam -f BAM -g hs -n sample --outdir peaks/

Narrow Peaks (TF, H3K4me3, H3K27ac)

macs3 callpeak \
    -t chip.bam \
    -c input.bam \
    -f BAM \
    -g hs \                        # hs=human, mm=mouse, ce=worm, dm=fly
    -n sample_narrow \
    --outdir peaks/ \
    -q 0.05                        # q-value threshold

Broad Peaks (H3K36me3, H3K27me3, H3K9me3)

macs3 callpeak \
    -t chip.bam \
    -c input.bam \
    -f BAM \
    -g hs \
    -n sample_broad \
    --outdir peaks/ \
    --broad \                      # Broad peak mode
    --broad-cutoff 0.1             # Broad peak q-value

Paired-End Data

# MACS2 uses BAMPE format for paired-end
macs3 callpeak \
    -t chip.bam \
    -c input.bam \
    -f BAMPE \                     # Paired-end BAM
    -g hs \
    -n sample_pe \
    --outdir peaks/

Multiple Replicates

# Pool replicates (MACS2 handles internally)
macs3 callpeak \
    -t rep1.bam rep2.bam rep3.bam \
    -c input.bam \
    -f BAM \
    -g hs \
    -n pooled \
    --outdir peaks/

Custom Genome Size

# For non-model organisms or custom genomes
macs3 callpeak \
    -t chip.bam \
    -c input.bam \
    -f BAM \
    -g 2.7e9 \                     # Effective genome size in bp
    -n sample \
    --outdir peaks/

Common Genome Sizes

Genome	Flag	Effective Size
Human	hs	2.7e9
Mouse	mm	1.87e9
C. elegans	ce	9e7
D. melanogaster	dm	1.2e8

Fixed Fragment Size

# If modeling fails or for ATAC-seq
macs3 callpeak \
    -t chip.bam \
    -c input.bam \
    -f BAM \
    -g hs \
    --nomodel \                    # Skip model building
    --extsize 200 \                # Fixed extension size
    -n sample \
    --outdir peaks/

Generate Signal Tracks

# Generate bedGraph and bigWig files
macs3 callpeak \
    -t chip.bam \
    -c input.bam \
    -f BAM \
    -g hs \
    -n sample \
    --outdir peaks/ \
    -B \                           # Generate bedGraph
    --SPMR                         # Signal per million reads

# Convert to bigWig (requires UCSC tools)
sort -k1,1 -k2,2n peaks/sample_treat_pileup.bdg > peaks/sample.sorted.bdg
bedGraphToBigWig peaks/sample.sorted.bdg chrom.sizes peaks/sample.bw

Local Lambda for Broad Marks

# Recommended for very broad marks
macs3 callpeak \
    -t chip.bam \
    -c input.bam \
    -f BAM \
    -g hs \
    --broad \
    --nolambda \                   # Use local lambda only
    -n sample \
    --outdir peaks/

Cutoff Analysis

# Test different q-value cutoffs
macs3 callpeak \
    -t chip.bam \
    -c input.bam \
    -f BAM \
    -g hs \
    --cutoff-analysis \            # Generate cutoff analysis file
    -n sample \
    --outdir peaks/

Output Files

File	Description
*_peaks.narrowPeak	Peak coordinates (BED6+4)
*_peaks.broadPeak	Broad peak coordinates
*_summits.bed	Peak summit positions
*_model.r	R script for model visualization
*_treat_pileup.bdg	Treatment signal (with -B)
*_control_lambda.bdg	Control signal (with -B)

narrowPeak Format

chr1  100  200  peak_1  100  .  5.2  10.5  8.3  50

Columns: chr, start, end, name, score, strand, signalValue, pValue, qValue, peak

Filter Peaks

# Filter by q-value
awk '$9 > 2' peaks.narrowPeak > peaks.filtered.narrowPeak  # -log10(q) > 2 means q < 0.01

# Sort by signal strength
sort -k7,7nr peaks.narrowPeak > peaks.sorted.narrowPeak

Key Parameters

Parameter	Default	Description
-t	required	Treatment BAM file(s)
-c	none	Control BAM file(s)
-f	AUTO	Format (BAM, BAMPE, BED)
-g	hs	Genome size
-n	NA	Output prefix
-q	0.05	Q-value cutoff
-p	none	P-value cutoff (overrides -q)
--broad	false	Broad peak calling
--nomodel	false	Skip model building
--extsize	200	Extension size (with --nomodel)
-B	false	Generate bedGraph
--SPMR	false	Signal per million reads

Related Skills

• peak-annotation - Annotate peaks to genes
• differential-binding - Compare peaks between conditions
• alignment-files - Prepare BAM files
• chipseq-visualization - Visualize peaks