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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. Use when calling peaks from ChIP-seq alignments. 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

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

# For MACS2 (legacy), replace 'macs3' with 'macs2' - syntax is identical

Without Input Control

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

Narrow Peaks (TF, H3K4me3, H3K27ac)

Copymacs3 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)

Copymacs3 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

Copy# MACS3 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

Copy# Pool replicates (MACS3 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

Copy# 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

Copy# 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

Copy# 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

Copy# 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

Copy# 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

Copychr1  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

Copy# 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