bio-long-read-sequencing-clair3-variants

name	bio-long-read-sequencing-clair3-variants
description	Calls germline small variants (SNPs and indels) from Oxford Nanopore and PacBio HiFi long reads with Clair3, a two-stage (pileup + full-alignment) deep-learning caller, selecting the chemistry- and basecaller-version-matched model, enabling read-based phasing, and benchmarking against GIAB with stratification. Covers why the model string is the experiment (no auto-detection, silent degradation on mismatch), why ONT homopolymer/STR indels are the residual error whole-genome F1 hides, and the somatic/trio/RNA boundary to the ClairS/Clair3-Trio family. Use when calling germline SNVs/indels from ONT or HiFi BAMs, choosing a Clair3 model, phasing variants, or benchmarking long-read calls.
tool_type	cli
primary_tool	Clair3

Version Compatibility

Reference examples tested with: Clair3 2.0+, whatshap 2.0+, bcftools 1.19+, hap.py 0.3.15+.

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags

Results depend on inputs that outlive the binary version - record them:

• The Clair3 MODEL must match the platform + chemistry + basecaller tier + basecaller version (e.g. r1041_e82_400bps_sup_v500). There is NO auto-detection; --model_path is mandatory and a mismatch silently degrades calls.
• Clair3 v2 moved TensorFlow -> PyTorch; models are pileup.pt/full_alignment.pt. v1 TensorFlow models do NOT load in v2.
• The full ONT model set (every version, hac/fast, _with_mv signal-aware) lives in the rerio clair3_models/ repo; only a subset is bundled.

If code throws an error, introspect the installed tool (run_clair3.sh --help) and adapt the example to the actual API rather than retrying.

Clair3 Variant Calling

"Call variants from my long reads" -> Run Clair3 with the model that matches how the reads were basecalled, phase, and benchmark with stratification - because the model string, not the command, determines accuracy.

• CLI: run_clair3.sh --bam_fn=aln.bam --ref_fn=ref.fa --output=out/ --threads=16 --platform=ont --model_path=/models/r1041_e82_400bps_sup_v500

Scope: germline diploid SNPs + small indels. NOT structural variants (-> structural-variants), NOT somatic/mosaic (-> ClairS/ClairS-TO), NOT RNA (-> Clair3-RNA).

The Single Most Important Modern Insight -- The Model String Is the Experiment, and ONT Indels Hide in the Strata

Clair3's accuracy is gated by two facts a naive user misses:

1. The model is hand-picked and a mismatch fails silently. There is no auto-detection - the user must point --model_path at a specific model folder. Three axes must ALL match: chemistry (r941 vs r1041), basecaller tier (fast/hac/sup), and basecaller version (g5014/v430/v500/v520), plus the optional _with_mv signal-aware axis if the BAM has Dorado mv tags. Wrong model = no crash, no warning, measurably worse calls (indels most). Derive the model from the basecaller string in the run metadata; pick the model version closest to but not above the basecaller version.
2. ONT indels in homopolymers/STRs are the residual error whole-genome F1 conceals. Even on R10.4.1 sup, insertions/deletions in homopolymer runs and short tandem repeats are the weak point (G/C homopolymers worst), because the pore cannot reliably count identical consecutive bases. A genome-wide indel F1 of ~99.5% hides much lower performance inside LowComplexity/homopolymer strata - exactly the medically relevant loci. HiFi largely solves this; do not transfer ONT-indel pessimism to HiFi. Always benchmark with GIAB stratification, never a single global number.

Two-Stage Architecture

Clair3 "symphonizes" two networks: a fast pileup model (summarized per-position statistics) that calls the large majority of sites, and a slow full-alignment model (haplotype-resolved read tensor) that re-evaluates only the uncertain subset. Internally Clair3 phases the top het-SNP pileup calls with WhatsHap, haplotags the BAM, and feeds the haplotagged reads to the full-alignment model - which is why read-based phasing buys ~6% indel F1, not cosmetics. Output: merge_output.vcf.gz (final).

Model Selection

Model name anatomy (r1041_e82_400bps_sup_v500): pore (r1041=R10.4.1), flowcell (e82), speed (400bps), basecaller tier (sup/hac/fast), basecaller version (v500=Dorado 5.0.0, g5014=Guppy 5.0.14). The _with_mv suffix uses Dorado move-table tags for best accuracy when present.

Data	--platform	Model
ONT R10.4.1 sup, Dorado v5.x, mv tags present	ont	r1041_e82_400bps_sup_v520_with_mv
ONT R10.4.1 sup, Dorado v5.0.0	ont	r1041_e82_400bps_sup_v500
ONT R10.4.1 hac	ont	r1041_e82_400bps_hac_v500/_v520
ONT R9.4.1 (any tier)	ont	r941_prom_sup_g5014
PacBio HiFi Revio	hifi	hifi_revio
PacBio HiFi Sequel II	hifi	hifi_sequel2
Illumina (supported)	ilmn	ilmn
PacBio CLR	-	not supported -> PEPPER-Margin-DeepVariant

Decision Tree by Scenario

Scenario	Tool	Why
Germline SNV/indel, single sample	Clair3	this skill
Somatic, paired tumor-normal	ClairS	VAF-aware; Clair3 germline priors cannot find low-VAF somatic
Somatic, tumor-only	ClairS-TO	tumor-only ensemble
De novo / Mendelian trio	Clair3-Nova / Clair3-Trio	family-aware
Long-read RNA variants	Clair3-RNA	RNA model
ONT R10.4.1, also considering DeepVariant	either	neck-and-neck on R10 sup; native-ONT DeepVariant (Kolesnikov 2024) superseded PEPPER-Margin
Non-human / draft / bacterial reference	Clair3 + --include_all_ctgs	default calls only chr1-22,X,Y -> empty output otherwise
Cohort joint genotyping	Clair3 gVCF -> GLnexus	bcftools merge on gVCFs is NOT joint genotyping

Core Commands

# Germline ONT calling (model MUST match the basecaller)
run_clair3.sh \
  --bam_fn=aln.bam --ref_fn=ref.fa --output=clair3_out/ \
  --threads=16 --platform=ont \
  --model_path=/opt/models/r1041_e82_400bps_sup_v500
# final VCF: clair3_out/merge_output.vcf.gz

# Phase the final output VCF (WhatsHap); --longphase_for_phasing swaps only the INTERNAL
# phaser to LongPhase (faster, SV-aware). For a LongPhase-phased final VCF use
# --use_longphase_for_final_output_phasing instead of --enable_phasing.
run_clair3.sh ... --enable_phasing --longphase_for_phasing
# Phased calls go to clair3_out/phased_merge_output.vcf.gz; merge_output.vcf.gz stays UNPHASED.

# Non-human / draft assembly reference - call ALL contigs
run_clair3.sh ... --include_all_ctgs

# Targeted / amplicon panel
run_clair3.sh ... --bed_fn=panel.bed --gvcf

# Benchmark against GIAB with stratification (the step that reveals ONT indel errors)
hap.py giab_truth.vcf.gz clair3_out/merge_output.vcf.gz \
  -f giab_confident.bed -r ref.fa --engine=vcfeval \
  --stratification giab_stratifications.tsv -o bench/hg002

Per-Method Failure Modes

Silent model mismatch

Trigger: --model_path pointing at a model that does not match the basecaller chemistry/tier/version. Mechanism: no auto-detection; the wrong network runs. Symptom: no error, lower F1 (indels most). Fix: derive the model from the basecaller string; verify the folder exists (rerio for the full set); for v2 ensure .pt models.

Clair3 found nothing on a non-human reference

Trigger: bacterial genome or draft assembly without chr1-22,X,Y names. Mechanism: Clair3 calls only standard human contigs by default. Symptom: near-empty VCF. Fix: --include_all_ctgs.

Global F1 looks great, clinical genes are wrong

Trigger: reporting only whole-genome F1. Mechanism: ONT indel errors concentrate in homopolymer/STR/low-complexity strata. Symptom: ~99.5% global indel F1 but much lower in LowComplexity. Fix: stratify with GIAB BEDs (Dwarshuis 2024); use CMRG for medically relevant genes.

Treating Clair3 as a somatic caller

Trigger: lowering --snp_min_af/--indel_min_af to catch low-VAF variants. Mechanism: germline model expects ~0.5/1.0 allele fractions, is not VAF-aware. Symptom: germline-model false positives at low AF, missed true somatic. Fix: ClairS (paired) / ClairS-TO (tumor-only).

v1 model with v2 Clair3

Trigger: an old TensorFlow model dir with Clair3 v2. Mechanism: v2 needs PyTorch .pt models. Symptom: model load failure. Fix: use pileup.pt/full_alignment.pt models (Converted Rerio).

Quantitative Thresholds

Threshold	Source	Rationale
Recommended depth ~20-60x	Clair3 guidance	sensitivity (hets, indels) falls off below ~20x; --min_coverage default 2 is a floor, not a recommendation
Phasing buys ~6% indel F1	Zheng 2022	haplotagged reads disambiguate indel alleles in repeats
ONT R10.4.1 sup: SNP F1 ~99.99%, indel F1 ~99.5%	GIAB benchmarks	indel residual lives in homopolymer/STR strata
--var_pct_full 0.3 (default)	Clair3 README	fraction of low-quality pileup calls re-run by full-alignment; raise for recall, slower
Stratify with GIAB / CMRG	Dwarshuis 2024	global F1 hides the ONT indel problem

Common Errors

Error / symptom	Cause	Solution
Empty/near-empty VCF on non-human ref	default calls only chr1-22,X,Y	--include_all_ctgs
Model fails to load	v1 TF model with v2 Clair3	use .pt (PyTorch) models
--model_path .../models/ont not found	no generic ont/hifi model	point at a specific model subfolder
Worse-than-expected indels	wrong-version or wrong-tier model	match the basecaller model exactly
"joint genotyping" gave odd merges	bcftools merge on gVCFs is not joint calling	use GLnexus
Looking for somatic/low-VAF variants	germline caller	use ClairS / ClairS-TO

References

• Zheng Z, Li S, Su J, Leung AWS, Lam TW, Luo R. 2022. Symphonizing pileup and full-alignment for deep learning-based long-read variant calling (Clair3). Nat Comput Sci 2:797-803.
• Zheng Z, He M, Yu X, et al. 2026. Accelerated long-read variant calling with Clair3 for whole-genome sequencing. Bioinformatics (advance access) btag181.
• Kolesnikov A, Cook D, Nattestad M, et al. 2024. Local read haplotagging enables accurate long-read small variant calling. Nat Commun 15:5907.
• Dwarshuis N, Kalra D, McDaniel J, et al. 2024. The GIAB genomic stratifications resource for human reference genomes. Nat Commun 15:9029.
• Lin JH, Chen LC, Yu SC, Huang YT. 2022. LongPhase: an ultra-fast chromosome-scale phasing algorithm for small and large variants. Bioinformatics 38(7):1816-1822.
• Chen L, Zheng Z, Su J, et al. 2025. ClairS-TO: a deep-learning method for long-read tumor-only somatic small variant calling. Nat Commun 16:9630.

Related Skills

• basecalling - The basecaller model+version the Clair3 model must match
• long-read-alignment - Produces the BAM (keep --MD; use minimap2 >=2.28)
• haplotype-phasing - whatshap/longphase phasing and haplotagging Clair3 uses internally
• medaka-polishing - ONT consensus; medaka diploid variant calling is deprecated in favor of Clair3
• structural-variants - SVs are out of Clair3's scope (Sniffles2/cuteSV)
• variant-calling/deepvariant - DeepVariant native ONT/HiFi models (neck-and-neck on R10)
• variant-calling/vcf-statistics - Summarize/filter the VCF Clair3 emits
• clinical-databases/variant-prioritization - Prioritize the called variants