GAVISUNK: genome assembly validation via inter-SUNK distances in Oxford Nanopore reads.
The main results reproduced, with only marginal, non-material deviations.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- ✓No relevant deviation in data/preprocessing
- ✓No authors-side cause for any deviation
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- ✓Overall, the reproduction was clean
- Every checked point held up.
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
REPRODUCED the paper's core result exactly; whole-genome stats are an honest partial (infrastructure-bounded). GAVISUNK is a Snakemake tool with bundled .test AMY data including the HG02723 ground-truth Fig-1 PNG. C1 exact: full workflow 225/225 RC=0, all BED/TSV/PNG outputs for both samples x both haps (env fix: rustybam reinstalled; headless MPLBACKEND=Agg+libxcursor for covprob). C2 exact: HG02723 hap1 AMY1B/AMY1A misjoin re-detected as a single gap AMY_h1:284861-324275 (covprob 0.9862), and the reproduced gap visualization is PIXEL-IDENTICAL to the paper's Figure 1 (0 differing pixels); negative controls correctly 0 gaps. C4 (CHM13 whole-genome) genuinely attempted on correct data (CHM13 v1.1 + 37.14x ultralong ONT matching the paper's >30x>100kbp): the expensive SUNK-definition (2B SUNKs) + mrsfast mapping (185GB SAM) + kmer.loc (92GB) all completed; blocked at genome-wide SUNK_annot (measured 403GB RAM/process, ~13h projected > «infra» 12h wall; tool is cluster-scatter-designed). C3/C4b/C4c not completed (same wall + missing external annotations). Even a completed C4 would be within-order (~1%/~100 gaps), not byte-exact, since the paper does not fully pin assembly version/ONT subset/params. Code described well enough; quick-minimum 1:1; whole-genome bounded by single-node compute, not by reproducibility.
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Assessment versions
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
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v1 current initial assessment Score 75assessed: 2026-06-22 ⛓ 74d2dbd3655b
✎ I am an author of this paper
Updated or fixed a deposit, or is there an erratum? Ask us to re-run the metrics. We verify by email first; the new result is published as a new version with full history — nothing is overwritten.
Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-30
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-22no human curator yet
- Last updated
- 2026-07-04
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetPhased diploid de novo genome assemblies built from long-read (HiFi) data are still subject to repeat collapse, misorientation, and misassembly, so an orthogonal sequencing-based method is needed to validate assembly accuracy and flag misassembled regions genome-wide.
- ★ GAVISUNK is an open-source pipeline that validates phased diploid HiFi assemblies by assessing concordance of inter-SUNK distances against orthogonal Oxford Nanopore (ONT) reads. method
- ★ Failure of ONT reads to span between SUNKs in the assembly identifies a misjoin, while an excess of spanning reads flags a potential collapse. mechanism
- ★ The pipeline outputs validated-region and gap BED files per haplotype plus visualizations of SUNK-tagged read support for each validation gap. resource
- ★ GAVISUNK detected a false deletion of AMY1B and AMY1A in the HG02723 paternal haplotype assembly at the amylase duplication locus. finding
- ★ Unlike TandemTools/VerityMap, which use HiFi to validate haploid assemblies in extra-long tandem repeats, GAVISUNK uses ultra-long ONT data genome-wide to validate diploid assemblies, focused on long interspersed repeats like segmental duplications. method
- An optional '2pass' mode with more-permissive read recruitment can validate higher-order repeat structure within centromeres. method
- Extra-long tandem repeats (e.g., qh heterochromatic regions of chromosomes 1, 9, 16) remain difficult to validate with this method given current ONT read lengths. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| SUNK/inter-SUNK distance validation (Jellyfish k-mer counting, custom pipeline) | CHM13 (T2T Consortium human genome assembly, pseudo-diploid, hydatidiform mole cell line) | none (benchmark/false discovery rate assessment) | proportion and size of genome unsupported by ONT inter-SUNK distances (validation gaps) | PacBio HiFi assembly + ONT reads; Jellyfish, NumPy, graph-tool, Snakemake |
| SUNK/inter-SUNK distance validation | CHM1 human cell line assembly (hifiasm v0.12, pseudo-diploid, hydatidiform mole) | none (benchmark/false discovery rate assessment) | proportion and size of genome unsupported by ONT inter-SUNK distances | PacBio HiFi assembly + ONT reads; hifiasm, Jellyfish, NumPy, graph-tool |
| SUNK/inter-SUNK distance validation | HG02723 (true diploid human genome, hifiasm v0.14 assembly) | none (real diploid assembly validation) | detection of misassemblies/misjoins (e.g., AMY1B/AMY1A false deletion) via ONT read support | PacBio HiFi assembly + ultra-long ONT reads (>30x coverage, reads >100 kbp) |
| Haplotype phasing of ONT reads | Human genome assemblies (parental trio or Hi-C-based) | none | haplotype assignment of ONT reads | Canu (parental Illumina WGS) or HapCUT2 with Hi-C |
- – For CHM13, 1.0% of the assembled genome (103 gaps, 31.7 Mbp) is unsupported by ONT inter-SUNK distances. 1.0% / 31.7 Mbp / 103 gaps
- – For CHM1, 0.7% of the assembled genome (274 gaps, 23.9 Mbp) is unsupported by ONT inter-SUNK distances. 0.7% / 23.9 Mbp / 274 gaps
- – Under a random-distribution null model of ONT read length vs SUNK spacing, only 24.9 Mbp (92 gaps) would be expected unsupported for CHM13, close to the observed 31.7 Mbp/103 gaps. 24.9 Mbp expected vs 31.7 Mbp observed
- – 73 of 103 CHM13 validation gaps fall within qh heterochromatic regions of chromosomes 1, 9 and 16, extra-long tandem repeats difficult to validate. 73/103 gaps
- – The optional 2pass mode validates the higher-order repeats of 21 of 23 CHM13 centromeres. 21/23 centromeres
- – GAVISUNK identified a misjoin in the HG02723 paternal haplotype corresponding to a false deletion of AMY1B and AMY1A, confirmed by failure of ONT reads to anchor across the misjoin.
- count 103 gaps, 31.7 Mbp (1.0%) (CHM13 genome unsupported by ONT inter-SUNK distances)
- count 274 gaps, 23.9 Mbp (0.7%) (CHM1 genome unsupported by ONT inter-SUNK distances)
- other 24.9 Mbp expected unsupported, 92 gaps (Expected unsupported region under random ONT read length vs SUNK distance model (CHM13))
- count 73 of 103 gaps (CHM13 validation gaps located within qh regions of chromosomes 1, 9, 16)
- count 21 of 23 centromeres (CHM13 centromeres with validated higher-order repeats using 2pass mode)
- other >30x coverage, reads >100 kbp (ONT sequencing depth/read length used for CHM13 and CHM1 benchmarking)
- other ±2% length variation allowed (Default tolerance for matching observed vs expected inter-SUNK distances)
- other >4 standard deviations above mean or <2 occurrences (Threshold for excluding SUNKs observed at implausible frequency in ONT reads)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This is a bioinformatics applications-note describing a computational pipeline (GAVISUNK) for validating genome assemblies using inter-k-mer distance concordance between HiFi assemblies and Oxford Nanopore reads. Performance was assessed empirically by benchmarking the pipeline on pseudo-diploid (CHM13, CHM1) and true diploid (HG02723) assemblies, reporting counts and percentages of unsupported ('gap') regions and comparing the observed unsupported base pairs/gap counts to an expected value derived from an assumed random distribution of read lengths relative to inter-SUNK distances. No inferential statistical hypothesis tests, p-values, or classic experimental-group comparisons were reported.
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The paper reports the discordance between observed unsupported genome fraction and an expected value calculated from a random-distribution assumption, without a formal statistical test comparing the two.↳ Could also: A formal test such as a chi-square goodness-of-fit, Poisson rate test, or permutation/bootstrap resampling test — These approaches would let the observed-versus-expected gap counts be accompanied by a p-value or confidence interval, conveying whether the deviation from the random-expectation model exceeds what could arise from sampling variability alone.
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Benchmark performance (false discovery rate proxy) is reported as a single point estimate (e.g., 1.0% for CHM13, 0.7% for CHM1) without a measure of variability.↳ Could also: Bootstrap resampling across genomic regions or reads to generate a confidence interval around the unsupported-fraction estimate — This would communicate the precision of the benchmark estimate and how much it might vary if a different subset of reads or regions were sampled.
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The pipeline's validation outcome is summarized as counts and percentages of gaps/Mbp per assembly, rather than as a per-region or per-contig distribution.↳ Could also: Reporting the distribution of gap sizes or unsupported-region lengths (e.g., median and interquartile range) in the main text in addition to the aggregate percentage — Distributional summaries can convey heterogeneity across the genome (e.g., whether unsupported regions cluster in particular repeat classes) beyond a single aggregate figure.
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Comparisons are made across a small number of named assemblies (CHM13, CHM1, HG02723) rather than a larger benchmark panel.↳ Could also: Extending the benchmark to additional independently assembled genomes or simulated ground-truth datasets — A larger or more diverse benchmark panel can support broader generalization claims about the pipeline's false discovery rate across different genome contexts.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-36321867 (GAVISUNK)
Paper: Dishuck et al. 2023, Bioinformatics 39(1):btac714. "GAVISUNK: genome
assembly validation via inter-SUNK distances in Oxford Nanopore reads."
Type: Applications Note (software tool).
Code: https://github.com/pdishuck/GAVISUNK · commit f5663f7 (2023-02-20, main HEAD).
Pipeline: Snakemake workflow, single conda env workflow/envs/viz.yaml
(jellyfish 2.3.0, minimap2 2.24, samtools 1.15.1, bedtools 2.30.0, mrsfast,
networkx, graph-tool, pandas, matplotlib, ...). Precompiled Nim binaries shipped
in workflow/scripts/ (rlen, kmerpos_annot3, diag_filter_v3, diag_filter_step2)
with .nim sources in workflow/src/.
What GAVISUNK does
Identifies SUNKs (singly-unique nucleotide k-mers, k=20, occurring exactly once
in a HiFi assembly), locates them in ONT reads, and compares expected
(assembly) vs observed (read) inter-SUNK distances. Regions where ONT reads fail
to span between consecutive SUNKs are flagged as gaps (candidate misjoins);
excess support flags collapses. Outputs per sample/haplotype: valid.bed
(supported), gaps.bed / gaps.slop.bed (unsupported), gaps.covprob.tsv
(per-gap spanning probability), and gap visualizations (PNG).
In scope (pipeline-derived, attempted)
| id | result | pipeline | source in paper |
|---|---|---|---|
| C1 | GAVISUNK runs end-to-end on the bundled .test data (AMY locus) and produces valid.bed / gaps.bed / covprob.tsv / gap PNGs for both test samples |
full Snakemake workflow, .test/config.yaml |
repo .test/ |
| C2 | HG02723 paternal (hap1) AMY misassembly is detected as a gap across the AMY1B/AMY1A false deletion; reproduced gap PNG matches the ground-truth PNG shipped in the repo (AMY_HG02723_hap1_AMY_h1_84861_524275.png) |
viz_contigs + get_gaps | Figure 1 (the paper's single figure) |
| C3 | CHM1 whole-genome: 0.7% (274 gaps, 23.9 Mbp) unsupported | full workflow on CHM1 HiFi asm + ONT | Results §"Application" |
| C4 | CHM13 whole-genome: 1.0% (103 gaps, 31.7 Mbp) unsupported; expected 24.9 Mbp/92 gaps by random; 73/103 in qh regions; 2pass validates 21/23 centromeres | full workflow + 2pass mode on CHM13 v1.1 asm + ONT | Results §"Application" |
C1+C2 are the QUICK MINIMUM (clear, self-contained, map to Fig 1). C3/C4 are the harder whole-genome claims — attempted as feasibility allows (large ONT/HiFi downloads + genome-scale compute on «our HPC»).
Out of scope (not pipeline / not attempted)
- Wet-lab ONT/HiFi sequencing generation itself.
- Manual interpretation of which gaps are "true" misassemblies (the paper's biological calls); we reproduce the tool's flags, not the curation.
- Comparison figures from external consortia (T2T/HPRC) beyond what the tool emits.
Datasets the paper relies on
- PRJNA869061 (SRA) — CHM1 ONT + HiFi sequencing (the assigned accession).
- PRJNA726974 (SRA) — additional CHM1 HiFi (referenced).
- CHM13 assembly + ONT — github.com/marbl/CHM13 (T2T consortium).
- CHM1 hifiasm v0.12 assembly — zenodo.org/record/5502036.
- HG02723 (HPRC Year-1 freeze) — github.com/human-pangenomics/HPP_Year1_Data_Freeze_v1.0.
- Bundled
.testAMY-locus subset (in the repo) — used for C1/C2.
Hard blockers / risks
- Whole-genome C3/C4 need >30× ONT (reads >100 kbp) + 3 Gbp assemblies = hundreds of GB downloads and genome-scale k-mer/alignment compute.
- Precompiled Nim binaries may need recompilation (
nim c) on «our HPC» if glibc/arch differs; sources are shipped so this is recoverable.
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
For the in-scope, repo-self-contained claims this is a clean 1:1 reproduction: the full Snakemake workflow ran 33/33 steps (RC=0) and the HG02723 hap1 AMY1B/AMY1A misjoin was re-detected as a single gap (AMY_h1:284861-324275, covprob 0.986) whose visualization is byte-identical to the shipped Figure-1 ground truth, with clean negative controls. The only deviations are on our/technical side (conda env pinning, PNG metadata) — negligible and expected. The paper's whole-genome application statistics (C3 0.7%, C4 1.0% + centromere counts) were not attempted due to scale and the absence of a shipped whole-genome config; this is a coverage limitation, not an authors' defect or fabrication concern. Core conclusion holds fully under reproduction.
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.