ATAC-seq Peak Calling

ATAC-seq Peak Calling

Basic MACS3 for ATAC-seq

# Standard ATAC-seq peak calling
macs3 callpeak \
    -t sample.bam \
    -f BAMPE \
    -g hs \
    -n sample \
    --outdir peaks/ \
    -q 0.05 \
    --nomodel \
    --shift -75 \
    --extsize 150 \
    --keep-dup all \
    -B

Key ATAC-seq Parameters

# Explained parameters
macs3 callpeak \
    -t sample.bam \        # Treatment BAM
    -f BAMPE \             # Paired-end BAM (uses fragment size)
    -g hs \                # Genome size: hs (human), mm (mouse)
    -n sample \            # Output name prefix
    --nomodel \            # Don't build shifting model
    --shift -75 \          # Shift reads to center on Tn5 cut site
    --extsize 150 \        # Extend reads to this size
    --keep-dup all \       # Keep duplicates (ATAC has natural duplicates)
    -B \                   # Generate bedGraph for visualization
    --call-summits         # Call peak summits

Why These Parameters?

Paired-End vs Single-End

# Paired-end (recommended for ATAC)
macs3 callpeak -f BAMPE -t sample.bam ...

# Single-end (less common)
macs3 callpeak -f BAM -t sample.bam \
    --nomodel --shift -75 --extsize 150 ...

Call Peaks on NFR Only

# First, filter to nucleosome-free reads (<100bp fragments)
samtools view -h sample.bam | \
    awk 'substr($0,1,1)=="@" || ($9>0 && $9<100) || ($9<0 && $9>-100)' | \
    samtools view -b > nfr.bam

# Call peaks on NFR
macs3 callpeak \
    -t nfr.bam \
    -f BAMPE \
    -g hs \
    -n sample_nfr \
    --nomodel \
    --shift -37 \
    --extsize 75 \
    --keep-dup all \
    -q 0.01

Broad Peaks (Optional)

# For broader accessible regions
macs3 callpeak \
    -t sample.bam \
    -f BAMPE \
    -g hs \
    -n sample_broad \
    --nomodel \
    --shift -75 \
    --extsize 150 \
    --broad \
    --broad-cutoff 0.1

Batch Processing

#!/​bin/​bash
GENOME=hs  # hs for human, mm for mouse
OUTDIR=peaks

mkdir -p $OUTDIR

for bam in *.bam; do
    sample=$(basename $bam .bam)
    echo "Processing $sample..."

    macs3 callpeak \
        -t $bam \
        -f BAMPE \
        -g $GENOME \
        -n $sample \
        --outdir $OUTDIR \
        --nomodel \
        --shift -75 \
        --extsize 150 \
        --keep-dup all \
        -q 0.05 \
        -B \
        --call-summits
done

Output Files

narrowPeak Format

chr1  100  500  peak1  500  .  10.5  50.2  45.1  200

Columns: chrom, start, end, name, score, strand, signalValue, pValue, qValue, summit_offset

Convert to BigWig

# Sort bedGraph
sort -k1,1 -k2,2n sample_treat_pileup.bdg > sample.sorted.bdg

# Convert to BigWig
bedGraphToBigWig sample.sorted.bdg chrom.sizes sample.bw

Merge Replicates

# Pool BAMs before peak calling (recommended for final peaks)
samtools merge -@ 8 merged.bam rep1.bam rep2.bam rep3.bam

# Call peaks on merged
macs3 callpeak -t merged.bam -f BAMPE -g hs -n merged ...

IDR for Replicate Consistency

# Call peaks on each replicate
macs3 callpeak -t rep1.bam -f BAMPE -g hs -n rep1 ...
macs3 callpeak -t rep2.bam -f BAMPE -g hs -n rep2 ...

# Run IDR
idr --samples rep1_peaks.narrowPeak rep2_peaks.narrowPeak \
    --input-file-type narrowPeak \
    --output-file idr_peaks.txt \
    --plot

# Filter by IDR threshold
awk '$5 >= 540' idr_peaks.txt > reproducible_peaks.bed

Related Skills

• read-alignment/​bowtie2-alignment - Align ATAC-seq reads
• atac-seq/​atac-qc - Quality control
• chip-seq/​peak-calling - ChIP-seq comparison
• genome-intervals/​bed-file-basics - Work with peak files