FridrichMethod Version Compatibility
Reference examples tested with: MACS3 3.0.4+, MACS2 2.2.9+, HOMER 4.11+, SPP 1.16+, samtools 1.19+, bedtools 2.31+, IDR 2.0.4+.
Before running, verify versions: <tool> --version and <tool> --help to confirm flags. If a flag is missing, check the changelog — MACS2->MACS3 is API-compatible for callpeak but predictd, bdgpeakcall, and hmmratac differ.
ChIP-seq Peak Calling
"Identify protein-DNA binding sites from ChIP-seq alignments" -> Detect statistically enriched genomic regions by comparing IP signal to input control (or genomic background), with peak shape (narrow/broad) determined by target biology (TF vs histone mark).
- CLI (ENCODE TF default):
macs2 callpeak -t chip.bam -c input.bam -f BAM -g hs -n sample --keep-dup all -p 1e-2 - CLI (ENCODE histone default): same with
--broad --broad-cutoff 0.1for H3K27me3, H3K9me3, H3K36me3 - CLI (alternative):
macs3 callpeak ...(API-identical, active development), HOMERfindPeaks tags/ -style histone -i input_tags/, SPP via phantompeakqualtools wrapper
ENCODE TF pipeline still uses SPP for peak ranking + IDR, with MACS2 producing the signal tracks. Histone pipeline uses MACS2 + naive overlap (IDR is too conservative for histone signal dynamic range). MACS3 is the actively maintained successor; MACS2 receives only bug fixes.
Critical Pre-Call Validation
Before any peak calling, three things must be true or the output is unreliable:
- Antibody validated — KO/KD orthogonal control, peptide-array specificity for histone modifications, or vendor-provided CRISPR-validated lot (Epicypher, CST). "ChIP-grade" marketing is not validation. See chipseq-qc.
- Fragment-size distribution is sane — TF ChIP should show sub-nucleosomal (
50-100 bp) enrichment; histone ChIP should show clean mono- (150) and di-nucleosomal (~300) peaks. Flat distribution = over-sonication; rescue is impossible. Check viasamtools view -f 0x2 sample.bam | awk '{print $9}' | sort | uniq -c. - Input control matches — Sonicated input is biased toward open chromatin; MNase input toward nucleosomes. Input from a different library prep batch or fragmentation method introduces bias that subtraction cannot fix.
Algorithmic Taxonomy
| Tool | Model | Treats fragments as | Strength | Fails when |
|---|---|---|---|---|
| MACS3/MACS2 callpeak | Dynamic local Poisson (max of genome-wide, 1kb, 5kb, 10kb lambda) + BH-FDR | Single-end shifts; PE fragments via BAMPE | Mature, fast, ENCODE-default, narrow + broad modes, integrated signal tracks | Confounds NFR with broad accessible domains; default narrow mode segments broad enrichment; assumes most genome NOT enriched (breaks for genome-wide marks) |
| SPP (Kharchenko 2008) | Strand cross-correlation peak detection + Poisson fold-enrichment | Single-end with cross-corr-derived shift | ENCODE TF caller; integrated NSC/RSC QC; robust for sharp TF peaks | Underperforms for broad marks; older R codebase; phantompeakqualtools wrapper has R-version compatibility issues |
HOMER -style factor |
Fixed-width peaks + three sequential filters (control / local / clonal) | Tag positions; auto-estimated width | Fast on tag directories; clonal filter -C removes PCR-artifact peaks |
Less calibrated p-values; fixed width clips variable-width factor binding |
HOMER -style histone |
Variable-width region stitching (500 bp blocks, 1000 bp gap merging); L=0 (no local enrichment) | Tag positions | Captures variable-width histone enrichment; Omnipeak benchmark (Shpynov & Artyomov 2026): outperforms -style factor for histone marks including H3K4me3 |
Less sensitive than MACS for very sharp TF binding |
Genrich -y (ChIP mode) |
q-value on log-transformed p-value, joint replicate model | Whole fragments (PE intervals) | Joint replicate analysis; chrM exclusion via -e chrM; auto blacklist via -E |
Less peer-reviewed than MACS/SPP; thin literature; control handling less mature |
| MACS3 hmmratac | 3-state HMM on fragment-size signal | Fragment-size classes | Best for ATAC, not ChIP | Wrong tool for ChIP; ChIP fragment-size distribution doesn't drive useful HMM states |
| SEACR (Meers 2019) | Empirical threshold on signal block totals | Bedgraph signal blocks | Designed for sparse CUT&RUN/CUT&Tag data; "stringent" mode with IgG strongly preferred | Not for traditional ChIP-seq (assumes near-zero background); see cut-and-run-tag |
| LanceOtron (Hentges 2022) | CNN trained on ENCODE peaks | bigWig signal | Competitive for both narrow and broad without parameter tuning | Newer; less validated; web-only or pip install |
For CUT&RUN / CUT&Tag specifically, see chip-seq/cut-and-run-tag — protocol differences (lower depth, IgG-only control, E. coli spike-in carryover) drive different caller choice (MACS2 + SEACR consensus, not MACS3 alone).
Decision: Narrow vs Broad
Driven by target biology, not preference. Calling broad mode does not make a sharp signal broad; it changes how MACS stitches adjacent enrichment.
| Target | Mode | Why |
|---|---|---|
| Transcription factors (CTCF, p53, GATA1, FOXA1) | Narrow (default) | Discrete motif binding produces sharp peaks |
| H3K4me3, H3K27ac at promoters/enhancers | Narrow | Localized at regulatory elements |
| H3K4me1 at enhancers | Narrow or broad-cutoff 0.1 | Variable; check published data for the cell type |
| H3K36me3, H3K79me2 (elongation) | Broad | Deposited across active gene bodies (5-50 kb domains) |
| H3K27me3, H3K9me3 (repressive) | Broad | Spread across 10-100+ kb domains |
| H4K20me3 (constitutive het) | Broad | Heterochromatin domains |
| Pol II (RNAPII) | Narrow at promoter + broad option for elongation profile | Two separate analyses if doing elongation biology |
For HOMER: use -style histone for ALL histone marks (Omnipeak benchmark, Shpynov & Artyomov 2026 NAR 54:gkaf1454); -style factor ONLY for transcription factors.
Decision: Model vs --nomodel
MACS2/3 fragment-size modeling needs ≥100 paired plus/minus enrichment regions within --mfold (default [5, 50]). Silent failure produces wrong fragment size and warped peaks — always inspect _model.r output.
| Condition | Model? | Fallback |
|---|---|---|
| Whole-genome, ≥1M treatment reads, narrow TF | Yes | --mfold 3 50 if fails |
Paired-end with -f BAMPE |
N/A | Fragment size from mate pairs |
| Single chromosome or targeted capture | No | --nomodel --extsize <data-derived or mark default> |
| Low read count (<500k) | No | Same |
| Broad histone mark | Either | Mark-type default if no estimate available |
When --nomodel is required, choose --extsize in priority order: (1) cross-correlation estimate from phantompeakqualtools (ENCODE standard, gives NSC/RSC simultaneously); (2) macs3 predictd -i chip.bam -g hs and read stderr; (3) mark-type fallback (147 for nucleosome-proximal marks, 200 for broader marks).
Effective Genome Size — Often Wrong, Always Matters
-g hs (2.7e9) and -g mm (1.87e9) are decade-old approximations. Modern read-length-matched values (deepTools effectiveGenomeSize table):
| Genome | Read length | Effective size |
|---|---|---|
| hg38 | 50 bp | 2.701e9 |
| hg38 | 75 bp | 2.748e9 |
| hg38 | 100 bp | 2.806e9 |
| hg38 | 150 bp | 2.862e9 |
| mm10 | 50 bp | 2.308e9 |
| mm10 | 100 bp | 2.467e9 |
Wrong size shifts every q-value but rarely peak ranks. For subset data (single chromosome, targeted), provide numeric -g <bp>; the shorthand inflates lambda_BG by 60× and produces false positives at low-signal regions.
Hyper-ChIPable Regions Are a Persistent Artifact
Teytelman 2013 (PNAS) and Park 2013 (PLoS One) demonstrated that highly-transcribed genes (rRNA, tRNA, histone gene cluster, snoRNA hosts, mitochondrial-encoded genes, abundant housekeeping loci) appear "bound" in ChIP-seq with untagged GFP, no antibody, or non-existent targets. ENCODE blacklist v2 catches repeat-driven artifacts but NOT these hyper-ChIPable transcribed regions.
Always interpret peaks at rRNA loci, tRNA clusters, replication-dependent histone genes (HIST1/2 clusters), mitochondrial DNA, and the top-1% input-signal regions with skepticism. For rigorous claims: (1) require motif enrichment at the peak (artifact has no motif); (2) require KO/KD signal loss; (3) build a cell-type-specific blacklist from the top 1% of input signal and intersect-out.
Pipeline Reference: ENCODE TF vs Histone
TF pipeline (uses SPP for peak ranking):
# Per-replicate (loose) — IDR tightens downstream
macs2 callpeak -t rep1.tagAlign.gz -c input.tagAlign.gz \
-f BED -g hs -n rep1 \
--nomodel --shift 0 --extsize {fraglen_from_xcor} \
--keep-dup all -B --SPMR -p 1e-2
# Repeat for rep2, pooled, and pseudoreplicates (split each rep into halves)
# Score peaks by signalValue, sort, run IDR (see Replicate Handling below)
Histone pipeline (uses MACS2 broad / narrow + naive overlap):
# Broad marks: H3K27me3, H3K9me3, H3K36me3
macs2 callpeak -t rep1.tagAlign.gz -c input.tagAlign.gz \
-f BED -g hs -n rep1 \
--broad --broad-cutoff 0.1 \
--nomodel --shift 0 --extsize {fraglen} \
--keep-dup all -B --SPMR -p 1e-2
# Naive overlap: a peak passes if it appears in ≥2 of N replicates
# with ≥40% reciprocal overlap (ENCODE default, often misquoted as 50%)
bedtools intersect -a rep1.broadPeak -b rep2.broadPeak -f 0.40 -r -u > naive_overlap.bed
--keep-dup all is intentional in the ENCODE pattern: duplicates were already filtered upstream by MarkDuplicates + samtools view -F 1804 -q 30. -p 1e-2 is permissive because IDR (TF) or overlap (histone) tightens downstream.
Replicate Handling: IDR vs Naive Overlap
ENCODE rules (Landt 2012 Genome Res):
TFs use IDR. Run on signal-ranked peaks (sort by -k8,8nr p-value; -k7,7nr signal works for SPP but breaks for MACS pile-up if libraries differ).
sort -k8,8nr rep1.narrowPeak > rep1.sorted
sort -k8,8nr rep2.narrowPeak > rep2.sorted
idr --samples rep1.sorted rep2.sorted \
--input-file-type narrowPeak --rank p.value \
--idr-threshold 0.05 --output-file true_reps.idr --plot
ENCODE Nself/Nt consistency rule (often misremembered):
- Nt = IDR-passing peaks across true biological replicates (threshold 0.05)
- Nself (per rep) = IDR-passing peaks across pseudoreplicates of one library (threshold 0.10)
- Library passes if
max(N1self, N2self) / min(N1self, N2self) ≤ 2ANDmax(Nt, max(Nself)) / min(Nt, min(Nself)) ≤ 2 - Both ratios > 2: library rejected
Histones use naive overlap. IDR's high-vs-low-rank assumption breaks for histone dynamic range. Naive overlap: pool peaks, require each to appear in ≥2 replicates with ≥40% reciprocal overlap.
ENCODE 3 vs ENCODE 4 Differences
| Feature | ENCODE 3 | ENCODE 4 |
|---|---|---|
| TF peak ranker | SPP | SPP (unchanged) |
| Histone caller | MACS2 | MACS2 (MACS3 not yet adopted) |
| Aligner | bwa-mem | bwa-mem (chromap evaluated; not yet swapped) |
| Blacklist | v1 (ENCODE DAC, unpublished resource) | v2 (Amemiya 2019) |
| TF significance | -p 1e-2 + IDR @ 0.05 |
Same |
| Histone significance | -p 1e-2 + naive overlap |
Same |
| Effective genome size | hs/mm shorthand |
deepTools read-length-tabulated |
| Pseudoreplicate IDR threshold | 0.10 self-consistency | 0.10 self-consistency |
ENCODE 4 outputs are NOT numerically comparable to ENCODE 3 on the same BAM (blacklist change + genome size update shift peak counts ~3-10%).
Per-Tool Failure Modes
MACS2/3 -- Silent fragment-size model failure
Trigger: Sparse signal, low replicate depth, or saturated samples; _model.r plot never inspected.
Mechanism: Model needs ≥100 paired plus/minus enriched regions in --mfold range. Below threshold, MACS picks an arbitrary fragment size (often 50 or 1000 bp), producing miscentered or oversized peaks. Stderr shows a warning that gets ignored.
Symptom: Peak summits shifted relative to known motif positions by hundreds of bp; visual inspection in IGV shows peaks displaced from pile-up centers.
Fix: Inspect <sample>_model.r — if peaks look reasonable, accept; if degenerate, widen with --mfold 3 50 or switch to --nomodel --extsize <data-derived>. For consistency across samples in a study, always use --nomodel --extsize {fraglen} with cross-correlation-derived fraglen (ENCODE pattern).
MACS2/3 -- Confounded narrow vs broad on intermediate marks
Trigger: Marks of intermediate breadth (H3K4me1, H3K9ac) called with default narrow mode.
Mechanism: Default narrow mode fragments wide enrichment into multiple sub-peaks; --broad over-stitches.
Symptom: Peak count 3-5× higher than published for same cell type; mean peak width < 200 bp at known enhancer regions.
Fix: For H3K4me1, try --broad --broad-cutoff 0.1 and compare; for H3K9ac, narrow mode typically OK. Always cross-reference published peak counts for the cell type and antibody lot.
MACS2/3 -- --call-summits double-counts
Trigger: Narrow mode + --call-summits flag.
Mechanism: MACS adds sub-peak summits at multi-mode pile-ups; broad-shouldered peaks get split into 2-3 entries.
Symptom: Peak count inflated; same genomic region appears as 2-3 adjacent peaks in narrowPeak output.
Fix: Drop --call-summits unless deliberately analyzing multi-mode binding (rare); merge bedtools merge -d 200 if needed post-hoc.
HOMER -- Wrong style for histones
Trigger: -style factor used for histone marks.
Mechanism: Factor mode uses fixed-width peaks with local enrichment filter -L 4 that eliminates broad signal.
Symptom: Far fewer peaks than expected for H3K4me3/H3K27ac/H3K27me3; missed enrichment at known regions.
Fix: Use -style histone for ALL histone marks (Omnipeak benchmark, Shpynov & Artyomov 2026); reserve -style factor for TFs only.
SPP / phantompeakqualtools -- R version incompatibility
Trigger: Running phantompeakqualtools wrapper script with R ≥ 4.0.
Mechanism: spp R package has unmaintained dependencies; some functions silently fail or return NaN for NSC/RSC.
Fix: Use conda env pinned to R 3.6 + spp 1.16; or use kundajelab/phantompeakqualtools fork (current); or substitute deepTools plotFingerprint for QC and MACS-derived fragment length.
chromap aligner -- Pre-applied shift double-counts
Trigger: Using chromap (fast aligner) output as MACS input with --shift -75 --extsize 150.
Mechanism: chromap pre-applies a Tn5/cut-site shift before fragment output (designed for ATAC); ChIP cut-site reasoning doesn't apply but the shift still happens silently.
Symptom: Peaks shifted ~5-10 bp from bwa-mem output at the same locus.
Fix: When using chromap, drop downstream shift OR use chromap's --no-correction. For ChIP, bwa-mem or bowtie2 are safer defaults until ENCODE switches.
Reconciliation: When Callers Disagree
| Pattern | Likely cause | Action |
|---|---|---|
| MACS finds peak; HOMER misses | HOMER local-enrichment filter (-L 4) removed it at low-signal regions; or -style factor clipped a histone peak |
Re-run HOMER with -style histone -L 0 for histones; if persists, trust MACS |
| HOMER finds peak; MACS misses | Clonal filter -C 2 retained PCR artifact peaks; or HOMER's auto-width captured something MACS narrow mode segmented |
Check if MACS broad mode rescues; check IGV for visual confirmation |
| SPP and MACS narrow peaks differ by 10-50 bp summit | Different fragment-size estimates (SPP uses cross-corr; MACS models from data) | Use same fragment size for both: ENCODE pattern --nomodel --extsize {xcor_fraglen} |
| MACS narrow + MACS broad on same data: 10× peak count difference | Expected — broad mode stitches subpeaks within 1 kb gap | Use narrow for differential analysis (consistent units); broad for domain annotation |
| Per-rep MACS calls peak; pooled MACS does not | One replicate dominates; pooling smooths local lambda | Trust pooled + IDR over per-replicate counts |
| Replicate count differs >2× | One replicate failed | Check FRiP, NSC, library complexity per replicate; do NOT average — drop the failing replicate or repeat |
Operational rule for publication-grade: TFs require IDR ≤ 0.05 on true reps AND Nt/Nself ratios ≤ 2. Histones require naive overlap ≥2 reps with ≥40% reciprocal overlap. Both require FRiP, NSC, RSC, and library complexity thresholds met. See chipseq-qc.
Common Errors
| Error / symptom | Cause | Solution |
|---|---|---|
| 0 peaks called | Wrong genome size on subset data; wrong -f for input format; swapped treatment/control |
Provide numeric -g; match -f to file type (BAM/BAMPE/BED); verify -t is enriched sample |
| Peak count >> 500k | Did not deduplicate; chrM not removed; -q too loose; hyper-ChIPable artifacts dominate |
Filter samtools view -F 1804 -q 30; remove chrM; tighten to -q 0.01; blacklist top-1% input regions |
| Peaks shifted from motif by ~75 bp | --shift not set for -f BAM; or fragment-size model wrong |
Add --shift 0 --extsize {fraglen}; or check _model.r |
--shift/--extsize ignored warning |
Used -f BAMPE with these flags |
Switch to -f BAM for ENCODE pattern, or accept that BAMPE uses true fragment spans |
| IDR returns 0 reproducible peaks | Sorted by wrong column; ranks effectively random | sort -k8,8nr (p-value descending) on each peakset |
| Naive overlap returns few peaks | Set -f 0.5 -r (50% reciprocal) — too strict |
Use -f 0.40 -r (ENCODE default) |
| FRiP < 1% | Bad ChIP (antibody, fragmentation, depth); peaks called on noise | Re-validate antibody with KO/KD; check fragment-size distribution; do not proceed |
References
- Park PJ 2009 Nat Rev Genet 10:669 (foundational review)
- Landt SG et al 2012 Genome Res 22:1813 (ENCODE/modENCODE guidelines, IDR Nself rule)
- Zhang Y et al 2008 Genome Biol 9:R137 (MACS)
- Kharchenko PV et al 2008 Nat Biotechnol 26:1351 (SPP)
- Heinz S et al 2010 Mol Cell 38:576 (HOMER)
- Li Q et al 2011 Ann Appl Stat 5:1752 (IDR framework)
- Teytelman L et al 2013 PNAS 110:18602 (hyper-ChIPable regions)
- Park D et al 2013 PLoS One 8:e83506 (independent hyper-ChIPable confirmation)
- Amemiya HM et al 2019 Sci Rep 9:9354 (ENCODE blacklist v2)
- ENCODE ChIP-seq pipeline v2.1.6 (github.com/ENCODE-DCC/chip-seq-pipeline)
- Shpynov O & Artyomov MN 2026 Nucleic Acids Res 54:gkaf1454 (Omnipeak; benchmarks HOMER -style histone vs factor for histone marks)
Related Skills
- chip-seq/chipseq-qc - Fragment-size diagnostic, FRiP, NSC/RSC, antibody validation
- chip-seq/cut-and-run-tag - SEACR + MACS for CUT&RUN/CUT&Tag (different QC, lower depth)
- chip-seq/spike-in-normalization - When global signal shifts expected (HDACi, BETi, EZH2i)
- chip-seq/differential-binding - DiffBind/csaw downstream of peak calling
- chip-seq/peak-annotation - Annotate peaks to genes and cCREs
- chip-seq/motif-analysis - Discover and scan binding motifs in peaks
- chip-seq/super-enhancers - Stitch H3K27ac peaks into super-enhancer calls
- atac-seq/atac-peak-calling - ATAC-specific shift/extend; no input control
- alignment-files/sam-bam-basics - Pre-call BAM filtering and deduplication
- genome-intervals/interval-arithmetic - Peak intersection and overlap