| name | bio-atac-seq-atac-peak-calling |
| description | Call accessible chromatin regions from ATAC-seq BAM files using MACS3, MACS2, Genrich, or HMMRATAC. Use when identifying open chromatin from aligned ATAC-seq, choosing between point-source vs HMM peak callers, applying ENCODE-style pseudoreplicate IDR, removing blacklist regions, or fixing 501bp consensus peaks for downstream differential analysis. |
| tool_type | cli |
| primary_tool | macs3 |
Version Compatibility
Reference examples tested with: MACS3 3.0.2+, MACS2 2.2.9+, Genrich 0.6.1+, HMMRATAC 1.2+ (now bundled in MACS3 as macs3 hmmratac), samtools 1.19+, bedtools 2.31+, IDR 2.0.4+.
Before using code patterns, verify installed versions match. If versions differ:
- CLI:
<tool> --version then <tool> --help to confirm flags
If code throws unexpected errors, introspect the installed binary (<tool> -h) and adapt the example to match the actual CLI rather than retrying.
ATAC-seq Peak Calling
"Call accessible regions from my ATAC-seq BAM" -> Identify Tn5-hypersensitive open chromatin, treating fragments as point insertion events (not protein-bound regions as in ChIP-seq) and accounting for the lack of input control.
- CLI (canonical, ENCODE 4):
macs2 callpeak -t atac.bam -f BAMPE -g hs -n sample --nomodel --shift -75 --extsize 150 --keep-dup all -B --SPMR -p 0.01
- CLI (HMM-based, single sample):
macs3 hmmratac -i atac.bam -n sample --outdir hmm_out
- CLI (joint replicates):
Genrich -j -t rep1.bam,rep2.bam -o peaks.narrowPeak -e chrM -E blacklist.bed
The -p 0.01 (loose) plus IDR is the ENCODE pattern: low stringency increases peak overlap between replicates, and IDR rescues the reproducible set. Single-sample workflows usually swap to -q 0.05 instead.
Algorithmic Taxonomy
| Tool | Model | Treats fragments as | Min reps | Strength | Fails when |
|---|
| MACS3/MACS2 | Local Poisson lambda + FDR | Point-source insertions (+/- shift) | 1 | Mature, ENCODE-default, fast, narrow + broad modes | Confounds NFR with broad accessible domains; no input means lambda from local genome only |
| Genrich (ATAC mode -j) | q-value on log-transformed p-value, joint replicate model | Whole fragments (paired-end intervals) | 1 (multi-rep optional) | Treats reps jointly; can exclude chrM via -e chrM; auto blacklist via -E; PCR-dup removal via -r | Less peer-reviewed than MACS; thin literature; slow on deep libraries |
| MACS3 hmmratac (was HMMRATAC) | 3-state HMM (open / nucleosomal / background) on fragment-size signal | Fragment-size classes | 1 | Models nucleosome periodicity directly; differentiates NFR and flanking nucleosomes | Needs >= 30M de-duplicated nuclear reads; memory-hungry; slow; flat fragment distribution -> garbage HMM |
HOMER findPeaks -style dnase | Fixed window + fold-change cutoff | Tag positions | 1 | Convenient for downstream HOMER motif analysis | Less calibrated p-values than MACS; window-size sensitive |
| nf-core/atacseq | Wrapper (MACS2 by default) | Same as MACS2 | 1 | Reproducible Nextflow pipeline with QC built in | Only as good as the underlying caller |
Methodology evolves; verify the current ENCODE ATAC-seq Standards (encodeproject.org pipelines/atac-seq) before locking parameters. ENCODE 4 still defaults to MACS2 (not MACS3) at time of writing; macs3 callpeak is API-compatible for ATAC parameters but not yet the official ENCODE binary.
Shift-Extend vs BAMPE: The Critical Choice
Two valid ways to feed paired-end ATAC into MACS:
Pattern A (ENCODE / "single-end-ified"): -f BAMPE actually IGNORES --shift/--extsize. To activate them, use -f BAM and treat each end independently. ENCODE's pipeline uses -f BAM --shift -75 --extsize 150 to model each Tn5 cut as a 150 bp window centered on the insertion site, ignoring fragment lengths.
Pattern B (paired-fragment): -f BAMPE uses the full paired-end fragment span as the signal interval. Best when fragment lengths are biologically meaningful (e.g., NFR-only peak calling at 38/75 bp). In BAMPE mode, do NOT set --shift/--extsize (silently ignored, but confusing).
For most bulk ATAC, Pattern A matches ENCODE convention and is reproducible against published peak sets. Pattern B can be more sensitive at narrow regulatory elements but does not match ENCODE outputs.
Effective Genome Size
-g hs and -g mm are MACS shorthands for old defaults. Modern values:
| Genome | MACS shorthand | Actual mappable size | Source |
|---|
| hg38 | -g hs (2.7e9) | 2.913e9 (50bp k-mer), 2.747e9 (75bp), 2.701e9 (100bp) | deepTools effectiveGenomeSize |
| hg19 | -g hs (2.7e9) | 2.864e9 (50bp), 2.701e9 (100bp) | deepTools |
| mm10 | -g mm (1.87e9) | 2.652e9 (50bp), 2.467e9 (75bp), 2.407e9 (100bp) | deepTools |
| mm39 | none | 2.654e9 (50bp), 2.494e9 (100bp) | deepTools |
Wrong size shifts every q-value but rarely changes peak ranks. Use unique-kmers.py (khmer) or the deepTools tabulated values for exact sizes; the shorthand is a decade-old approximation.
Effective Genome Size: When It Matters
Trigger: Comparing peaks across genome builds or species; reproducing published q-value cutoffs; hi-resolution lambda estimation.
Mechanism: MACS estimates genome-wide lambda as total_reads / effective_size. Wrong size -> wrong null -> shifted q-values, especially at the marginal cutoff.
Symptom: Peak counts diverge ~10-20% from published numbers when re-running an old dataset.
Fix: Pull the read-length-matched value from deepTools effectiveGenomeSize table. For pipelines, parameterize this; never inline the shorthand for cross-study comparisons.
Per-Tool Failure Modes
MACS2/MACS3 -- Confounded NFR + broad accessibility
Trigger: Cell type with extended open domains (e.g., active super-enhancers, MYOD1 regulons, locus-control regions).
Mechanism: Default narrow-peak mode segments wide accessible domains into multiple smaller peaks at local lambda spikes; --broad --broad-cutoff 0.1 merges them but inflates total length and breaks IDR comparability.
Symptom: Peak count >> 200k for human bulk ATAC at ENCODE depth; mean peak width < 200 bp; visual inspection in IGV shows 3-5 calls under one continuous accessibility block.
Fix: Run both narrow and broad; use narrow for differential analysis, broad for domain-level enrichment (e.g., super-enhancer overlap). Do NOT use --call-summits for broad mode.
Genrich -- Replicate weighting and chrM exclusion
Trigger: Replicates with very different library sizes; high-mitochondrial samples not pre-filtered.
Mechanism: Genrich's joint mode pools reads via Fisher's method. Library-size imbalance dominates the joint p-value; chrM reads inflate background unless -e chrM is set.
Symptom: Most-significant peaks cluster on chrM or on the largest-library replicate's high-coverage regions.
Fix: Always pass -e chrM (Genrich 0.6+) and -E blacklist.bed. Down-sample BAMs to common depth (samtools view -s) before joint calling if libraries differ >2x. Add -r to remove PCR duplicates inside Genrich, OR pre-deduplicate (do not do both).
MACS3 hmmratac (HMMRATAC) -- Depth and fragment-size dependence
Trigger: Library < 25M nuclear reads, or libraries with degraded chromatin and flat fragment-size distribution.
Mechanism: The 3-state HMM is trained from fragment-size classes (NFR ~50 bp, mono ~200 bp, di ~400 bp peaks). Without periodicity the emission distributions collapse and the HMM cannot separate states.
Symptom: Output BED is empty, or all peaks are tiny (~150 bp) with no nucleosome flanks called; runtime explodes (>24h) on shallow data.
Fix: Verify fragment-size periodicity in QC first (atac-qc skill). If flat, fall back to MACS3 callpeak. HMMRATAC needs >= 30M deduplicated nuclear reads per ENCODE recommendation.
HOMER findPeaks -- Window-size sensitivity
Trigger: Default -style dnase uses 75 bp peaks; ATAC peaks are 250-500 bp typically.
Mechanism: HOMER's window-based caller does not auto-fit width to ATAC.
Fix: Use -style factor -size 150 for narrow ATAC peaks, or skip HOMER for peak calling and use it only for downstream motif analysis on MACS peaks.
Aligner choice -- chromap vs bwa-mem2 vs bowtie2 affects peak shape
Trigger: Switching aligners between datasets and expecting reproducible peaks.
Mechanism: chromap (Zhang 2021) applies its own ATAC-specific 4 bp / -5 bp Tn5 shift before fragment output; bwa-mem2 and bowtie2 do not. Downstream --shift -75 --extsize 150 parameters are calibrated for unshifted bwa/bowtie BAMs; applying them to chromap output double-shifts the signal.
Symptom: Peaks called from chromap output are shifted by ~5-10 bp relative to bwa output at the same locus.
Fix: When using chromap, drop --shift and --extsize (chromap's pre-shift is sufficient) OR use chromap's --no-correction flag to disable Tn5 shift and proceed with standard MACS parameters. Document the aligner version and any shift choices in methods. Within a project, pin the aligner.
Single-sample (no replicate) -- Rotation / circular-shift permutation
Trigger: Single biological sample without any replicate for IDR.
Mechanism: IDR requires two replicates by construction. For n=1, statistical confidence per peak comes from local background (Poisson p-value) but reproducibility cannot be assessed.
Fix: Apply a stricter -q 0.01 (vs ENCODE -p 0.01 + IDR pattern) and additionally apply rotation/circular-shift permutation: shift the BAM cuts by a random distance modulo each chromosome and re-call peaks; the per-peak persistence rate across rotations is a non-parametric reproducibility proxy. Document this is a single-sample heuristic, not ENCODE-compliant.
ENCODE 3 vs ENCODE 4 Differences
| Feature | ENCODE 3 (legacy) | ENCODE 4 (current) |
|---|
| Per-rep significance threshold | -q 0.05 directly | -p 0.01 (loose) + IDR |
| Pseudoreplicate IDR cutoff | Not formalized | --idr-threshold 0.10 self-consistency |
| TSS enrichment threshold | >= 6 (older) | >= 7 (hg38, GENCODE v29) |
| Mt fraction expectation | < 25% | < 20% (Omni-ATAC < 5%) |
| Blacklist | v1 | v2 (Amemiya 2019) |
| Default genome size | hardcoded hs/mm | encouraged: deepTools effectiveGenomeSize |
To reproduce a published ENCODE 3 dataset, pin the original pipeline and threshold exactly. ENCODE 4 results are not directly numerically comparable to ENCODE 3 even on the same input BAM.
Super-Enhancer Detection
For active super-enhancer (SE) annotation alongside narrow-peak workflow, ROSE (Whyte 2013) and LILY (Boeva 2017) stitch ATAC or H3K27ac peaks separated by < 12.5 kb and rank by signal:
ROSE_main.py -g hg38 -i atac_peaks.gff -r atac.bam -o rose_out/ -t 2500
ROSE-style stitching is complementary to MACS3 narrow peaks: narrow peaks for differential analysis; SE annotation for biology interpretation. SE calls require H3K27ac input for definitive annotation; ATAC alone produces "stretch enhancers" that overlap but are not identical to H3K27ac SE.
ENCODE 4 ATAC-seq Pipeline (Reference Implementation)
The exact ENCODE pattern produces the most-comparable peak sets:
macs2 callpeak \
-t rep1.filt.dedup.bam \
-f BAM -g hs \
-n rep1 --outdir peaks/rep1/ \
--nomodel --shift -75 --extsize 150 \
--keep-dup all \
-B --SPMR \
-p 0.01
macs2 callpeak \
-t rep1.filt.dedup.bam rep2.filt.dedup.bam \
-f BAM -g hs -n pooled --outdir peaks/pooled/ \
--nomodel --shift -75 --extsize 150 --keep-dup all -B --SPMR -p 0.01
samtools view -b -h -s 1.5 rep1.filt.dedup.bam > rep1.psr1.bam
samtools view -b -h -s 2.5 rep1.filt.dedup.bam > rep1.psr2.bam
--SPMR writes signal as Signal Per Million Reads (normalized bedGraph). -p 0.01 is intentionally loose; IDR will tighten to a reproducible set.
IDR for Reproducible Peaks
Goal: Find peaks reproducible across biological replicates at controlled IDR.
Approach: Score paired peak lists by signalValue, fit IDR's two-component mixture (reproducible + noise), threshold at IDR <= 0.05 (true reps) or 0.10 (pseudoreplicates).
sort -k8,8nr rep1_peaks.narrowPeak > rep1.sorted.narrowPeak
sort -k8,8nr rep2_peaks.narrowPeak > rep2.sorted.narrowPeak
idr --samples rep1.sorted.narrowPeak rep2.sorted.narrowPeak \
--input-file-type narrowPeak --rank p.value \
--output-file true_reps.idr \
--idr-threshold 0.05 --plot --log-output-file idr.log
idr --samples psr1_peaks.narrowPeak psr2_peaks.narrowPeak \
--input-file-type narrowPeak --rank p.value \
--output-file psr.idr --idr-threshold 0.10 --plot
ENCODE consistency rules: Nt = peaks passing IDR on true reps; Nself = peaks passing IDR on pseudoreps. Library passes if max(Nt, Nself) / min(Nt, Nself) <= 2. If both ratios > 2, the library is rejected.
IDR fails when: Ranking column choice matters. --rank p.value (column 8) is robust; --rank signal.value (column 7) breaks if MACS pile-up scaling differs between replicates.
Decision Tree by Experimental Scenario
| Scenario | Recommended caller | Why |
|---|
| Bulk ATAC, 2-3 reps, depth >= 25M | MACS2 ENCODE pipeline + IDR | Reproducible, comparable to published peaksets |
| Bulk ATAC, 1 sample (no rep) | MACS3 callpeak with -q 0.05; do not run IDR | IDR is meaningless without reps; tighter q-value substitutes |
| Bulk ATAC, depth >= 30M, want NFR + flanking nuc structure | MACS3 hmmratac | HMM separates NFR from nucleosome flanks |
| Multi-replicate joint analysis where rep weighting is symmetric | Genrich -j ATAC mode | Joint p-value across reps; built-in chrM and blacklist |
| Cell type with broad super-enhancer accessibility | MACS3 --broad --broad-cutoff 0.1 for SE; narrow for differential | Domain-level inference vs site-level |
| FFPE / degraded chromatin (flat fragment dist) | MACS3 callpeak with stringent -q 0.01; never HMMRATAC | HMM needs fragment periodicity |
| scATAC pseudobulk per cluster | MACS3 callpeak per cluster + iterative overlap | See atac-seq/single-cell-atac |
| Want fixed-width consensus peaks for differential | Call broadly, then re-center to summits +/- 250 bp | See atac-seq/consensus-peakset |
| Plant / non-model organism | MACS3 with -g <effective_size>; verify size empirically | Default -g hs/mm invalid; compute via khmer |
Reconciliation: When Callers Disagree
| Pattern | Likely cause | Action |
|---|
| MACS narrow peaks much fewer than Genrich | Genrich q-cutoff different default (-q 0.05 log-scale, MACS -q 0.05 linear) | Re-run with -q 0.01 (Genrich) for parity |
| HMMRATAC misses peaks MACS finds | Library too shallow OR fragment-size periodicity weak | Trust MACS; HMMRATAC is depth-sensitive |
| HMMRATAC calls peaks MACS misses | HMM is sensitive to mid-strength accessibility flanked by phased nucleosomes | Inspect; often genuine but unconfirmed by short-fragment signal |
| Same peak called by all but width 2x different | Broad mode vs narrow mode mismatch | Standardize: re-center to summit +/- 250 bp for differential |
| Per-rep MACS calls peak; pooled MACS does not | One rep dominates; lambda smoothes it out in pooled | Trust pooled + IDR over per-rep counts |
Operational rule for high-confidence reporting: Require a peak to pass IDR <= 0.05 on true replicates AND survive blacklist/greylist filtering AND have mean signalValue >= 5 across reps. Two callers from different families (MACS + Genrich) agreeing within 250 bp is acceptable evidence when IDR is unavailable.
Blacklist and Greylist
wget https://github.com/Boyle-Lab/Blacklist/raw/master/lists/hg38-blacklist.v2.bed.gz
gunzip hg38-blacklist.v2.bed.gz
bedtools intersect -v -a peaks.narrowPeak -b hg38-blacklist.v2.bed > peaks.no_blacklist.narrowPeak
Blacklist is mandatory; greylist is optional and most useful when the same library prep produces consistent artifact regions across samples.
NFR-Only Peak Calling
Goal: Call peaks using only sub-nucleosomal fragments (<100 bp) for sharper TF-binding-relevant accessibility.
Approach: Pre-filter BAM to short fragments, then call peaks with parameters scaled to the smaller fragment length.
samtools view -h sample.dedup.bam | \
awk 'substr($0,1,1)=="@" || ($9 > 0 && $9 < 100) || ($9 < 0 && $9 > -100)' | \
samtools view -b > nfr.bam
samtools index nfr.bam
macs2 callpeak -t nfr.bam -f BAM -g hs -n sample_nfr \
--nomodel --shift -37 --extsize 75 \
--keep-dup all -p 0.01
--shift -37 --extsize 75 halves both parameters to match shorter fragments; this is what TOBIAS recommends for footprinting input.
Output Files (narrowPeak)
| Column | Field | Notes |
|---|
| 1-3 | chrom, start, end | 0-based, half-open |
| 4 | name | MACS auto-numbers |
| 5 | score | Min(int(-10*log10(qvalue)), 1000) |
| 6 | strand | . for ATAC |
| 7 | signalValue | Fold enrichment over local lambda |
| 8 | pValue | -log10 p |
| 9 | qValue | -log10 q (BH-FDR) |
| 10 | summit_offset | Peak summit relative to start |
Convert to bigWig for browsers: sort -k1,1 -k2,2n sample_treat_pileup.bdg | bedGraphToBigWig - chrom.sizes sample.bw.
Common Errors
| Error / symptom | Cause | Solution |
|---|
--shift/--extsize ignored warning | Used -f BAMPE with these flags | Switch to -f BAM or remove the flags |
| 0 peaks called | Forgot --nomodel; MACS tries to build a shifting model and fails | Add --nomodel --shift -75 --extsize 150 |
| Peak count >> 500k | Did not deduplicate; or did not remove chrM; or -q too loose | Pre-filter (samtools view -F 1804 -q 30; samtools idxstats); use -q 0.01 |
Sequence chrM not found (Genrich) | Wrong chromosome name in -e flag (chrM vs MT) | Match BAM header naming convention |
| HMMRATAC out of memory | -Xmx heap too small; HMMRATAC defaults to 4G | Increase heap: java -Xmx16g -jar HMMRATAC.jar ... |
| Peaks shifted by 75 bp from expected positions | Forgot --shift -75 (cuts at one end of read) | Add the shift; positions are now centered on Tn5 cut site |
| IDR returns 0 reproducible peaks | Sorted by wrong column; ranks are random | Sort each peakset by -k8,8nr (p-value descending) |
References
- Buenrostro JD et al 2013 Nat Methods 10:1213 (ATAC-seq protocol)
- Corces MR et al 2017 Nat Methods 14:959 (Omni-ATAC, fixed-width peaks)
- Tarbell ED & Liu T 2019 Nucleic Acids Res 47:e91 (HMMRATAC)
- Gaspar JM 2018 bioRxiv 459545 (Genrich)
- Li Q et al 2011 Ann Appl Stat 5:1752 (IDR framework)
- Landt SG et al 2012 Genome Res 22:1813 (ENCODE/modENCODE peak calling guidelines, IDR Nself rule)
- Amemiya HM et al 2019 Sci Rep 9:9354 (ENCODE blacklist v2)
- ENCODE ATAC-seq Standards (encodeproject.org/atac-seq) -- canonical pipeline parameters
Related Skills
- atac-seq/atac-qc - Verify TSS enrichment, FRiP, and fragment periodicity before calling
- atac-seq/consensus-peakset - Combine per-sample peaks into a fixed-width differential-ready set
- atac-seq/single-cell-atac - Pseudobulk peak calling per cluster
- atac-seq/differential-accessibility - Downstream DiffBind/csaw/DESeq2 testing
- atac-seq/deep-learning-atac - chromBPNet bias-corrected per-base profiles as alternative input
- read-alignment/bowtie2-alignment - Upstream ATAC alignment
- alignment-files/duplicate-handling - Pre-call dedup with Picard MarkDuplicates
- chip-seq/peak-calling - ChIP-seq comparison (uses input control)
- chip-seq/super-enhancers - ROSE / LILY for super-enhancer annotation
- genome-intervals/bed-file-basics - Peak file manipulation
