GPTomics/bio-long-read-sequencing-structural-variants

Version Compatibility

Reference examples tested with: Sniffles 2.2+, cuteSV 2.1+, minimap2 2.28+, samtools 1.19+, truvari 4.0+.

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 reference-matched tandem-repeat BED supplied to the caller (Sniffles --tandem-repeats) drives the FP rate in repeats more than any other setting. Record which TR BED was used.
• Benchmark numbers depend on the region set + TR handling + Truvari params; record all three.
• cuteSV parameters are platform-specific (ONT vs HiFi vs CLR); the defaults are not platform-appropriate.

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

Long-Read Structural Variants

"Find structural variants in my long reads" -> Map with the SV-ready preset (soft-clipped supplementaries), call with a TR-aware caller, and benchmark stating the region set and Truvari params.

• CLI: sniffles --input aln.bam --vcf svs.vcf --reference ref.fa --tandem-repeats TR.bed

Long reads are the killer app for SVs: a single read spans the breakpoint (within-read CIGAR or split alignment) and resolves repeats short reads cannot. By convention SV = >=50 bp; the 30-100 bp range is a VNTR-dominated gray zone where callers disagree most.

The Single Most Important Modern Insight -- An SV Call Is a Representation Artifact as Much as a Biological Fact

In tandem repeats and segmental duplications, the same biological event has many valid VCF encodings - a deletion can be written as the reciprocal insertion on the other allele, and a VNTR expansion's breakpoints slide freely across repeat units. Consequently:

1. The tandem-repeat BED, the aligner, and the Truvari parameters decide precision/recall as much as the caller does. A claim like "caller X has F1 0.95" is meaningless without also stating the region set, the TR BED supplied to the caller, and the Truvari params - change any one and the number moves more than the gap between callers.
2. Without a TR BED, one event fragments into several false-positive calls with inconsistent breakpoints. --tandem-repeats makes clustering repeat-aware (widening the merge window inside annotated TRs) - the single biggest FP-reduction lever, not a nicety.
3. truvari refine exists precisely to re-harmonize representations within TR regions; benchmarking TR-dense regions without it systematically understates recall.

Caller Taxonomy

| Tool | Regime | Best for | Citation | |------|--------|----------|----------| | Sniffles2 | germline + population + mosaic | the default germline workhorse; cohort joint genotyping; .snf merge | Smolka 2024 Nat Biotechnol 42:1571 | | cuteSV | germline | high sensitivity, speed; per-platform tuning required | Jiang 2020 Genome Biol 21:189 | | SVIM | germline | scores (not hard-filters) SVs; good INS detection | Heller 2019 Bioinformatics 35:2907 | | pbsv | germline (PacBio) | two-step discover->call; official PacBio tool | PacBio (no journal paper) | | NanoVar | germline, low-depth | 4-8x ONT clinical | Tham 2020 Genome Biol 21:56 | | dipcall / SVIM-asm / PAV | assembly-based germline | most accurate single sample with phased HiFi; truth-set generation | Li 2018; Heller 2021; Ebert 2021 | | Severus | somatic (tumor-normal) | cancer T/N, complex/subclonal | Keskus 2026 Nat Biotechnol | | nanomonsv | somatic (tumor-normal) | precise somatic breakpoints, MEI | Shiraishi 2023 NAR 51:e74 | | SVision-pro | de novo + somatic, complex | resolving nested CSVs | Wang 2025 Nat Biotechnol 43:181 |

Decision Tree by Scenario

| Scenario | Recommended | Why | |----------|-------------|-----| | Single ONT/HiFi germline sample | Sniffles2 + --tandem-repeats | TR-aware, auto support, fast | | Cohort germline | Sniffles2 per-sample .snf -> merge | re-genotypes from raw signal; true joint genotypes | | Maximum sensitivity / speed | cuteSV with the platform-matched param set | per-platform tuning is mandatory | | Phased HiFi, want best per-sample accuracy | assembly-based (dipcall/SVIM-asm) -> hifi-assembly | resolves the alt haplotype directly | | Tumor-normal somatic SVs | Severus or nanomonsv | paired callers; Sniffles --mosaic is single-sample only | | Low-VAF mosaic in one sample | Sniffles2 --mosaic | lowers support, reports VAF (not a T/N caller) | | Low coverage (4-8x) | NanoVar | designed for low-depth clinical | | Benchmarking | Truvari (+refine) vs GIAB Tier1/CMRG | the field standard; state region + params |

Alignment for SV Calling

Map with minimap2 (the modern default; NGMLR is a higher-precision/slower legacy niche for Sniffles). Use the platform preset and keep soft-clipped supplementary alignments - split-read callers reconstruct breakpoints from the clipped sequence on those records.

minimap2 -ax map-ont --MD -Y ref.fa ont.fq.gz | samtools sort -o aln.bam && samtools index aln.bam
#   -Y keeps SEQ on supplementaries (the SV substrate); --MD for cuteSV; map-hifi/map-pb for PacBio

Sniffles2 - germline and the .snf population workflow

# Single sample (always supply --reference for INS sequence and --tandem-repeats for repeats)
sniffles --input aln.bam --vcf svs.vcf --reference ref.fa --tandem-repeats human_GRCh38_TR.bed

# Cohort: per-sample .snf signature index, then merge + joint-genotype
sniffles --input s1.bam --snf s1.snf --reference ref.fa --tandem-repeats TR.bed
sniffles --input s2.bam --snf s2.snf --reference ref.fa --tandem-repeats TR.bed
sniffles --input s1.snf s2.snf --vcf cohort.vcf --reference ref.fa

# Force-call / regenotype a known SV set in a new sample
sniffles --input new.bam --genotype-vcf known_svs.vcf --vcf genotyped.vcf

# Single-sample low-VAF / mosaic (NOT a tumor-normal caller)
sniffles --input tumor.bam --vcf mosaic.vcf --mosaic

The .snf is a binary signature index (NOT a VCF - never bcftools it); it retains sub-threshold signatures so the merge re-genotypes an SV even in a sample that did not independently pass support.

cuteSV - the per-platform parameter trap

cuteSV's defaults are not platform-appropriate; the README gives distinct sets by error rate. --genotype is OFF by default. Positional args: cuteSV <bam> <ref> <out.vcf> <work_dir>. Force-calling moved to the separate cuteFC tool.

| Platform | --max_cluster_bias_INS | --diff_ratio_merging_INS | --max_cluster_bias_DEL | --diff_ratio_merging_DEL | |----------|------------------------|--------------------------|------------------------|--------------------------| | ONT | 100 | 0.3 | 100 | 0.3 | | PacBio HiFi/CCS | 1000 | 0.9 | 1000 | 0.5 | | PacBio CLR | 100 | 0.3 | 200 | 0.5 |

mkdir cutesv_work
cuteSV aln.bam ref.fa cutesv.vcf cutesv_work --genotype \
  --max_cluster_bias_INS 100 --diff_ratio_merging_INS 0.3 \
  --max_cluster_bias_DEL 100 --diff_ratio_merging_DEL 0.3   # ONT set

Benchmarking with Truvari

truvari bench --base giab_tier1.vcf.gz --comp calls.vcf.gz \
  --includebed tier1_regions.bed --pctseq 0.7 --refdist 500 --passonly -o bench/
truvari refine bench/        # re-harmonize TR-region representations for a fair comparison

--pctseq (default 0.7) compares the actual inserted/deleted sequence, not just coordinates - set 0 for depth-based callers lacking alt sequence, keep 0.7 for long-read callers. Region set dominates the headline: Tier1 (resolvable INS/DEL >=50 bp) overstates whole-genome performance; CMRG reflects hard clinical loci. Tier1 v0.6 is INS/DEL only - do not report INV recall against it.

Per-Method Failure Modes

One VNTR fragments into many false positives

Trigger: calling in tandem repeats without a TR BED. Mechanism: the breakpoint slides across repeat units, scattering signatures. Symptom: several calls with inconsistent breakpoints where one event exists. Fix: supply --tandem-repeats to the caller; truvari refine when benchmarking.

cuteSV defaults inflate or fragment calls

Trigger: running cuteSV with one parameter set across platforms. Mechanism: HiFi settings over-merge ONT noise; ONT settings fragment clean HiFi signatures. Symptom: FP inflation or split calls. Fix: use the platform-matched set; remember --genotype is off by default.

Missing insertion sequence / breakpoints

Trigger: Sniffles without --reference, or alignment without -Y. Mechanism: no reference -> no ALT sequence; hard-clipped supplementaries -> lost breakpoint sequence. Symptom: INS lack sequence; imprecise breakpoints. Fix: add --reference and align with -Y.

Treating Sniffles --mosaic as a cancer caller

Trigger: somatic SV calling with single-sample --mosaic. Mechanism: mosaic mode lowers support in one sample; it has no normal to subtract. Symptom: germline SVs reported as somatic; FP at low VAF. Fix: Severus or nanomonsv (paired tumor-normal).

Comparing F1 across studies that handled repeats differently

Trigger: quoting F1 without region + TR BED + Truvari params. Mechanism: representation handling moves the number more than the caller. Symptom: apples-to-oranges comparisons. Fix: fix the region set, TR BED, and Truvari params; run truvari refine.

Quantitative Thresholds

| Threshold | Source | Rationale | |-----------|--------|-----------| | SV >= 50 bp | GIAB convention | 30-100 bp is a VNTR gray zone where callers disagree | | Sniffles --minsvlen 35, --mapq 25, --minsupport auto | Sniffles2 manpage | the actual defaults (support is coverage-derived, not a fixed 3) | | Coverage ~20-30x germline; >30-60x mosaic/somatic | SV practice | large SVs callable from 5-10x; low-VAF needs depth | | Truvari --pctseq 0.7, --refdist 500 | English 2022 | sequence-aware INS matching; loosen refdist to 1000 only for fuzzy callers | | cuteSV params per platform | cuteSV README | error rate sets cluster bias / merge ratio |

Common Errors

| Error / symptom | Cause | Solution | |-----------------|-------|----------| | Many FP calls in repeats | no TR BED | supply --tandem-repeats | | cuteSV VCF has no GT | --genotype off by default | add --genotype | | Cannot bcftools the .snf | .snf is a binary signature index | use it as Sniffles input, not a VCF | | INS records lack sequence | --reference not supplied | add --reference ref.fa | | Imprecise/missing breakpoints | supplementaries hard-clipped | align with minimap2 -Y | | Looking for cuteSV force-calling flag | moved to cuteFC | use the cuteFC tool | | Somatic SVs from a single sample | germline/mosaic caller | Severus / nanomonsv (paired) |

References

• Smolka M, Paulin LF, Grochowski CM, et al. 2024. Detection of mosaic and population-level structural variants with Sniffles2. Nat Biotechnol 42:1571-1580.
• Jiang T, Liu Y, Jiang Y, et al. 2020. Long-read-based human genomic structural variation detection with cuteSV. Genome Biol 21:189.
• Heller D, Vingron M. 2019. SVIM: structural variant identification using mapped long reads. Bioinformatics 35:2907-2915.
• English AC, Menon VK, Gibbs RA, Metcalf GA, Sedlazeck FJ. 2022. Truvari: refined structural variant comparison preserves allelic diversity. Genome Biol 23:271.
• Zook JM, Hansen NF, Olson ND, et al. 2020. A robust benchmark for detection of germline large deletions and insertions. Nat Biotechnol 38:1347-1355.
• Wagner J, Olson ND, Harris L, et al. 2022. Curated variation benchmarks for challenging medically relevant autosomal genes (CMRG). Nat Biotechnol 40:672-680.
• Keskus AG, Bryant A, Ahmad T, et al. 2026. Severus detects somatic structural variation and complex rearrangements in cancer genomes using long-read sequencing. Nat Biotechnol 44:247-257.

Related Skills

• long-read-alignment - SV-ready mapping (-Y soft-clip, platform preset)
• basecalling - Read accuracy/length that gates breakpoint precision
• clair3-variants - Small variants (<50 bp) are Clair3's job, not an SV caller's
• haplotype-phasing - Haplotag the BAM for haplotype-specific / phased SVs
• genome-assembly/hifi-assembly - Phased assembly for assembly-based SV calling
• variant-calling/structural-variant-calling - The variant-calling-side SV view
• variant-calling/vcf-manipulation - Filter/merge the SV VCFs
• genome-intervals/gtf-gff-handling - Annotate SVs against gene models