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

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

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

Related Skills

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