Diminutive, degraded but dissimilar: Wolbachia genomes from filarial nematodes do not conform to a single paradigm.
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 authors-side cause for any deviation
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- 🟡A deviation arose in the data or preprocessing
- 🟡Overall, the reproduction showed a material discrepancy
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
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When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-18
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-18no human curator yet
- Last updated
- 2026-07-29
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: opusThe study tests whether Wolbachia genomes from filarial nematodes conform to a single paradigm of reductive genome evolution and co-evolutionary/symbiotic relationship with their hosts, using newly enriched and sequenced genomes spanning multiple supergroups.
- ★ wCtub and wDcau (863 988 bp and 863 427 bp) are the smallest Wolbachia genomes sequenced to date and are the first genomes representing supergroup J. finding
- ★ Supergroup J is a valid Wolbachia clade, confirmed by complete genomes despite prior controversy from MLST weaknesses. finding
- ★ Wolbachia genomes from filarial nematodes contain low levels of transposable elements and lack intact bacteriophage sequences, unlike many arthropod Wolbachia. finding
- ★ There is no single Wolbachia–filarial nematode pattern of co-evolution or symbiotic relationship. finding
- ★ Strong synteny exists between supergroup C and supergroup J Wolbachia, while supergroup D Wolbachia carry more transposable elements than other supergroups. finding
- ★ Metabolic pathway analysis shows highly conserved pathways (haem and nucleotide biosynthesis) and more variable pathways (vitamin B biosynthesis) that may be specific to certain host–symbiont associations. mechanism
- ★ A DNA-enrichment/target-capture method (LEFT-SEQ and Illumina capture using biotinylated probes) was used to produce four complete and two draft Wolbachia genomes from filarial nematodes. method
- ★ Six new Wolbachia genomes (wCtub, wDcau, wDimm, wLsig, wLbra, wMhie) representing supergroups C, D, F and J are provided as genomic resources. resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Target-enrichment PacBio long-read sequencing (LEFT-SEQ) | Wolbachia from Dirofilaria immitis, Litomosoides sigmodontis, Cruorifilaria tuberocauda, Dipetalonema caudispina (filarial nematodes) | none | Wolbachia genome sequence/assembly | PacBio Sequel; SMRTbell express template kit v2.0; biotinylated probes (Roche NimbleGen) |
| Target-enrichment Illumina sequencing (hybrid capture) | Wolbachia from Madathamugadia hiepei, Litomosoides brasiliensis, Dipetalonema caudispina (filarial nematodes) | none | Wolbachia genome sequence/assembly | Illumina; SeqCap EZ HyperCap (Roche NimbleGen); NEBNext Ultra II FS DNA kit |
| Whole-genome shotgun sequencing without capture (Illumina and PacBio) | Filarial nematode host DNA / Wolbachia | none | reads for assembly and host genome | Illumina; PacBio Sequel; SMRTbell express template prep kit v2.0; NEBNext Ultra II FS DNA library prep kit |
| De novo genome assembly and comparative genomics pipeline | Six Wolbachia strains (supergroups C, D, F, J) | none | genome size, gene content, transposable elements, synteny, metabolic pathways | Canu, Spades, Unicycler, Bowtie2, ngmlr, pear, blastn, QUAST, seqtk, Trim Galore! |
- – wCtub genome assembled at 863 988 bp 863 988 bp
- – wDcau genome assembled at 863 427 bp 863 427 bp
- ▼ Filarial Wolbachia genomes are smaller than arthropod Wolbachia genomes 957 990–1 080 084 bp (filarial) vs 1 250 060–1 801 626 bp (arthropod)
- ▲ Supergroup D Wolbachia show more transposable elements than other filarial supergroups
- – Strong synteny observed between supergroup C and supergroup J Wolbachia
- – No global co-evolutionary pattern detected among Wolbachia from filarial nematodes
- – Haem and nucleotide biosynthesis pathways are highly conserved while vitamin B biosynthesis is variable across strains
- count 863 988 bp (wCtub) (smallest Wolbachia genome size)
- count 863 427 bp (wDcau) (smallest Wolbachia genome size)
- other 84 % (proportion of available Wolbachia genomes belonging to supergroups A and B)
- count 24 complete and 55 draft genomes (Wolbachia genomes available to date)
- count 17 supergroups (A–F, H–Q, S) (Wolbachia phylogenetic lineages)
- other 2.5 % wsp divergence threshold (original arbitrary supergroup delimitation)
- count 4 complete + 2 draft genomes (new Wolbachia genomes produced in this study)
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 descriptive comparative genomics study sequencing and assembling six new Wolbachia genomes (four complete, two draft) from filarial nematodes using PacBio and Illumina sequencing with biotinylated-probe capture enrichment. Genome content, size, transposable element load, synteny, and metabolic pathway completeness were compared across Wolbachia supergroups using bioinformatics tools; no classical inferential hypothesis tests (e.g., t-tests, ANOVA) are applied in the excerpted text. Results are reported descriptively as genome statistics, BLAST similarity thresholds, and qualitative pathway-presence assessments.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| BLAST nucleotide similarity filtering (blastn, similarity >80 %, bitscore >50) | Identification and isolation of Wolbachia contigs from de novo assemblies | — | not stated |
| De novo genome assembly quality assessment (QUAST assembly statistics) | Evaluation of assembled Wolbachia genome contigs/scaffolds | — | na |
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Wolbachia contigs were identified by blastn with fixed similarity (>80 %) and bitscore (>50) thresholds, applied as hard filters without statistical framing↳ Could also: Average Nucleotide Identity (ANI, e.g., via fastANI or pyANI) or Mash distance could also be used to assess contig-level genomic relatedness to reference Wolbachia sequences — ANI and Mash provide continuous, genome-wide relatedness estimates that are less sensitive to the choice of a single similarity cutoff and are increasingly standard for prokaryote species-level classification
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Genome completeness was assessed via assembly statistics (QUAST) and manual curation↳ Could also: A marker-gene completeness tool such as BUSCO or CheckM could also be applied to estimate genome completeness against a conserved single-copy ortholog set — BUSCO/CheckM provide a standardized, reproducible completeness score widely used for reduced-genome endosymbionts, facilitating direct comparison across published datasets
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Metabolic pathway presence or absence was assessed qualitatively (pathway complete vs. incomplete)↳ Could also: A quantitative pathway coverage metric (e.g., fraction of pathway reactions encoded, as in KEGG completeness scoring or MinPath) could also be reported — A numeric completeness score allows graded comparison across genomes and supergroups rather than binary present/absent calls, which can obscure partial pathways of potential functional significance
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Transposable element content and other genomic features were compared descriptively across supergroups without formal statistical testing↳ Could also: A non-parametric test (e.g., Kruskal-Wallis or Mann-Whitney U) or permutation-based approach could also be used to assess whether TE load or other continuous genomic metrics differ significantly across supergroup categories — Even with small n, a formal test (with appropriate acknowledgement of low power) would distinguish observed differences from random variation and provide a reproducible effect-size estimate
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Synteny was assessed visually and qualitatively between supergroups↳ Could also: A quantitative synteny measure such as a genome rearrangement distance (e.g., breakpoint distance computed by tools like SyMAP, MUMmer, or Mauve with permutation testing) could also be reported — Quantitative rearrangement distances allow synteny conservation to be placed on a comparable scale across all pairwise genome comparisons, not only the highlighted C–J pair
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Phylogenetic relationships among Wolbachia strains are central to the study but the phylogenetic method details are not fully described in the available text excerpt↳ Could also: Whole-genome phylogenetic approaches (e.g., core-genome SNP phylogeny, GTDB-Tk placement, or concatenated single-copy ortholog trees with bootstrap support) could also complement or replace MLST-based trees — Whole-genome methods use far more informative sites than MLST loci, which the authors themselves note have weaknesses for resolving supergroup J; genome-wide approaches are now standard for endosymbiont phylogenomics
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-33295865 (Wolbachia genomes from filarial nematodes)
Paper: Lefoulon et al. 2020, Microb Genom, DOI 10.1099/mgen.0.000487. Code link in brief: https://github.com/lh3/seqtk — a generic FASTA/FASTQ toolkit, NOT the authors' own pipeline. Per BRIEF rule P16, applying an existing third-party tool to the paper's own data is equally valid. seqtk's role in the paper was adapter/ chimera removal in PacBio read pre-processing ("in-house shell script", not shipped).
Data: BioProject PRJNA593581. Raw reads SRR10903008–10, SRR10902913–14, SRR10900508–11, SRR10898805–06. Deposited assemblies (the reproducible artifacts):
- Wolbachia (6): wLsig GCA_013365435.1 (CP046577), wDimm GCA_013365455.1 (CP046578), wCtub GCA_013365475.1 (CP046579), wDcau GCA_013365495.1 (CP046580), wLbra GCA_013366805.1 (WQMO00000000), wMhie GCA_013366855.1 (WQMP00000000)
- Nematode hosts (6): nDimm GCA_013365355.1, nLsig GCA_013365315.1, nCtub GCA_013365365.1, nDcau GCA_013365325.1, nLbra GCA_013365375.1, nMhie GCA_013365335.1
In scope (pipeline-derived, reproducible)
The paper's central quantitative result is Table 3 (genome assembly statistics). These are recomputable EXACTLY from the deposited assemblies — a direct 1:1 check of the reported values and of "delivers-what-promised":
- Genome size (bp) — sum of sequence lengths (seqtk / QUAST). Deterministic.
- G+C content (mol%) — seqtk comp. Deterministic; paper gives 1–2 decimals, NCBI rounds to 0.5, so recomputing from FASTA is the value-add.
- Number of contigs — count of FASTA records (QUAST). Deterministic.
- N50 (bp) — QUAST. Deterministic.
- BUSCO completeness (%) — BUSCO v3, proteobacteria_odb9 (221 genes) for the 6 Wolbachia genomes; nematoda_odb9 (982 genes) for the 6 host genomes. Reproducible but tool-version-sensitive (paper used busco v3.0.2 / Augustus); treated as within-tol target, not byte-exact.
Out of scope (not attempted — explained)
- De novo assembly itself (Canu / SPAdes / Unicycler + blastn contamination filtering + minimus2 circularization + manual curation). Explicitly manual and unspecified ("in-house shell script", "manually curated to eliminate contaminations"). NOT reproducible 1:1; we instead verify the deposited products of this pipeline.
- ANI / dDDH (external web servers: ANI Calculator, GGDC) — wet-lab-analog web tools.
- RAST, ISsaga, PHASTER, Prokka, KASS annotation; OrthoFinder/Gblocks/IQ-TREE phylogenomics; MUMmer synteny; PACo/Parafit cophylogeny — large multi-tool chains, several via web servers, far beyond the 80% floor; deferred / not attempted.
Primary reproduction targets
Table 3, all 12 genomes × {size, GC, contigs, N50, BUSCO}. Wolbachia genomes first (the headline of the paper), then host genomes.
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.
This is a clean, near-1:1 reproduction of the paper's central quantitative result (Table 3 assembly statistics) recomputed directly from the deposited GCA assemblies: all 6 Wolbachia genomes reproduce exactly (24/24) and 4/6 host genomes are exact. The only genuine deviation is on the data-deposit side: nCtub and nDcau public deposits are one short contig smaller than the printed table, which makes nCtub GC mismatch (30.29 vs 29.70). This is a benign deposit-versioning artifact (likely post-deposit NCBI contamination screening), not a computation error, and shows no fabrication signal; the core 'diminutive, degraded but dissimilar' conclusion is fully confirmed.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
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