Manual curation for improved genome annotation of the functionally extinct northern white rhinoceros (Ceratotherium simum cottoni).
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.
- Nothing in this column.
- 🟡Could not use the authors’ exact input data
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡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
Described well enough to partially reproduce via the paper's OWN shipped annotation (P16). The repo (eruggeri/Northern-White-Rhinoceros-Annotation @2807cc3) ships only the final annotation as Git-LFS GTF/GFF3, no scripts (Galaxy workflow is a PDF). 1:1 result: 3 of 4 numeric Table-2 claims reproduce within a few percent when the curated subset is isolated from the file - transcripts 32975 vs 34385 (-4.1%), avg transcript length 37615 vs 36981 bp (+1.7%, and this confirms 'length' means genomic span incl. introns), genome coverage 49.7% vs 48% (genome 2,495,808,350 bp). Gene count partial: the file's ids mix SwissProt entry-names and HGNC symbols (23793 raw tokens) so 15738 is not cleanly recoverable without an external mapping DB. KEY finding for auditors: the shipped GTF/GFF3 is the FULL un-curated StringTie merged assembly (475,980 transcripts / 430k MSTRG genes) - a SUPERSET; Table 2 describes the curated subset embedded within it, so a naive feature count of the repo file gives ~14x the reported transcripts. No fabrication evidence: reported numbers are consistent with the curated subset of the shipped data. BUSCO (C5) NOT reproduced - BUSCO 5.8.2 transcriptome mode crashed with a runtime BatchFatalError (no aligner initialised); env built and 32267 transcripts extracted fine, so it is a tool bug not a data issue, and the paper never stated BUSCO version/lineage/mode anyway. NOT attempted (out of scope/hard-20%): the manual curation itself (subjective BLAST-based; the headline +81% genes depends on it) and re-running HISAT2+StringTie2 from raw GEO FASTQ (heavy, and would not reproduce post-curation Table-2 numbers).
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
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 69assessed: 2026-06-14 ⛓ 44e0e61fbaba
✎ 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
- 2026-06-14
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-15no human curator yet
- Last updated
- 2026-08-05
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: opusCan RNA sequencing of granulosa cells combined with de novo transcript assembly and extensive manual curation substantially improve the limited and error-prone genome annotation of the functionally extinct northern white rhinoceros (Ceratotherium simum cottoni)?
- ★ Manual curation of RNA-seq-derived de novo transcripts increased the number of functional genes in the NWR annotation by 81% (from 8,701 to 15,738). finding
- ★ The number of annotated functional transcripts increased by 141% (from 14,274 to 34,385) in the new annotation. finding
- ★ Using in vivo collected granulosa cells across developmental stages for RNA-seq and StringTie de novo assembly yields a more diverse, complete annotation. method
- ★ The improved annotation corrects gene nomenclature to HGNC/VGNC conventions and fixes erroneous, protein-named, and bacterial gene assignments, making it operational for transcriptional studies. resource
- ★ BUSCO analysis showed 93.4% completeness, supporting the quality and completeness of the transcriptome/annotation. finding
- The original BRAKER3-based annotation was flawed: only 51% of transcripts were called to the correct genetic sequence, with many protein-named, misassigned, or bacterial genes. finding
- Total transcript sequence length covers 48% of the genome assembly, consistent with total-RNA sequencing capturing unspliced transcripts, repeats, and possible pseudogenes. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| bulk RNA-seq (total RNA, stranded, Ribo-Zero) | southern white rhinoceros (Ceratotherium simum simum) in vivo collected mural granulosa cells; reads mapped to NWR genome CerSimCot1.0 | none | transcript reads for de novo transcript assembly and annotation | Illumina TruSeq Stranded Total RNA with Ribo-Zero Plus; 100 bp paired-end on Illumina NovaSeq 6000 (GSE261038, 6 samples) |
| bulk RNA-seq (total RNA, stranded, Ribo-Zero) | southern white rhinoceros granulosa cells; reads mapped to NWR genome CerSimCot1.0 | none | transcript reads for de novo transcript assembly and annotation | Illumina Stranded Total RNA with Ribo-Zero Plus (NovaSeq X plus prep); 150 bp paired-end on Illumina NovaSeq 6000 (GSE300824, 8 libraries) |
| RNA isolation and quality control | granulosa cells in RNAlater | none | RNA quantity and RNA integrity number (RIN > 6.0 threshold) | Arcturus PicoPure RNA Isolation Kit; Qubit 4 Fluorometer; Agilent 4150 TapeStation |
| read alignment and de novo transcript assembly | NWR genome CerSimCot1.0 (GCA_021442165.1) | none | aligned reads and merged transcript annotation file | Galaxy (usegalaxy.org); HISAT2; StringTie2 |
| manual curation via sequence homology search | de novo transcript sequences vs Ceratotherium simum simum, Diceros bicornis, Equus caballus | none | gene assignment at ≥80% percent identity; corrected nomenclature | NCBI BLAST+ |
| transcriptome completeness assessment | assembled NWR transcriptome | none | BUSCO completeness (single-copy, duplicated) | BUSCO / OrthoDB |
| transrectal ovum pickup (OPU) sample collection | four southern white rhinoceros females; ten follicles (2 growing, 6 dominant, 2 pre-ovulatory) | none | mural granulosa cells collected | ultrasound-guided probe with double-lumen needles |
- ▲ Functional genes increased from 8,701 to 15,738 in the new annotation 81% increase
- ▲ Functional transcripts increased from 14,274 to 34,385 141% increase
- – BUSCO completeness of the transcriptome 93.4% (single copy 89.4%, duplicated 3.9%)
- ▲ Percent of genome covered by exons rose from 1% to 48% 1% to 48%
- – Average transcript length remained essentially unchanged between annotations 36,981 bp vs 39,878 bp
- – Only 51% of original annotated transcripts were correctly called to genetic sequence 7,299/14,274 (51%)
- – Average sequencing depth per sample 37,364,052 reads
- – New gene count approaches other mammalian genomes (cow Ensembl release 113: 20,848 gene models) 15,738 vs 20,848
- count 15,738 functional genes (new) vs 8,701 (original) (annotated genes, 81% increase)
- count 34,385 transcripts (new) vs 14,274 (original) (annotated transcripts, 141% increase)
- other 93.4% (single copy 89.4%, duplicated 3.9%) (BUSCO completeness of transcriptome)
- mean 37,364,052 (average sequencing depth per sample across 14 samples)
- count 7,299/14,274 (51%) (original transcripts called to correct genetic sequence; 6,763 to protein names, 212 misassigned, 455 to bacterial genes)
- other 48% (percent of genome assembly covered by total transcript sequence/exons (vs 1% original))
- other ≥80% percent identity (BLAST threshold for assigning a sequence to a known gene)
- count 20,848 gene models (cow gene models, Ensembl release 113, for comparison)
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 methods paper describing the manual curation and improvement of the northern white rhinoceros genome annotation using RNA-seq from 14 granulosa cell samples collected from four southern white rhinoceros females. Reads were aligned with HISAT2 and assembled into transcripts with StringTie2 on the Galaxy platform, then manually curated via NCBI BLAST homology searches (≥80% nucleotide identity) against related species. All results are reported descriptively as raw counts and percentages comparing the original and new annotations; transcriptome completeness was assessed with BUSCO (93.4%). No inferential statistical tests were performed.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| BUSCO completeness assessment | Quality evaluation of the final curated transcriptome annotation | — | na |
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The average sequencing depth across 14 samples (37,364,052 reads) is reported without a measure of spread; individual values in Table 1 range from ~23 M to ~64 M reads↳ Could also: Report standard deviation or range alongside the mean sequencing depth — A dispersion measure would convey whether coverage was consistent across samples, which is relevant context for assessing whether lower-depth samples could have limited transcript discovery for the merged annotation
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Transcript assembly was performed with StringTie2 on each sample individually and then merged into a union annotation↳ Could also: Use a dedicated genome annotation pipeline such as MAKER2, Liftoff, or EVidenceModeler (EVM) to integrate RNA-seq evidence with protein homology evidence in a more automated framework — Structured annotation pipelines weight multiple evidence types systematically and produce standardized quality scores (e.g., annotation edit distance), which could reduce the manual curation burden and make the annotation process more reproducible
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Homology-based manual curation applied a single fixed 80% nucleotide identity threshold for gene assignment via BLASTn↳ Could also: Complement nucleotide BLAST with protein-level searches (BLASTx or BLASTp against UniProt/OrthoFinder) — Protein-level homology is more sensitive at typical evolutionary distances between rhinoceros and horse, and can identify conserved genes where nucleotide identity falls below the 80% threshold while the encoded protein remains recognisable
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Read alignment was performed with HISAT2↳ Could also: Use STAR (Spliced Transcripts Alignment to a Reference) as an alternative splice-aware aligner — STAR is widely benchmarked alongside HISAT2 and can differ in sensitivity for novel splice junctions, which matters when annotating a genome with limited prior transcript evidence; comparing aligners is a common quality-control step in annotation projects
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Annotation completeness was evaluated solely with BUSCO↳ Could also: Complement BUSCO with protein-level completeness tools such as OMArk, or with annotation consistency metrics such as annotation edit distance (AED) scores — BUSCO captures single-copy orthologue representation but does not assess isoform accuracy or gene-model structural quality; additional metrics provide complementary views of annotation completeness and correctness
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Granulosa cells across follicular developmental stages were the sole tissue source for transcript discovery↳ Could also: Incorporate RNA-seq from additional tissue types (e.g., blood, fibroblasts, liver) to broaden transcriptome coverage — Many genes are expressed in a tissue-restricted manner; multi-tissue sampling is a standard strategy in genome annotation projects to approach the full complement of expressed genes, which would likely increase the BUSCO completeness score beyond the 93.4% achieved here
Citation network
Where this publication sits in the reproducibility-weighted citation graph — what it is built on, and what is built on it. Citation data from OpenAlex.
No assessed neighbours yet — the network grows as more papers are assessed.
Data lineage
The datasets this paper uses (text-mined from the full text via Europe PMC), and which other assessed papers stand on the same data. A shared dataset is a factual link — not a judgement.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-41490125
Paper: Ruggeri et al. (PLoS One, 2026). Manual curation for improved genome annotation of the functionally extinct northern white rhinoceros. DOI 10.1371/journal.pone.0340594 · PMCID PMC12768360
Shipped artifacts: GitHub eruggeri/Northern-White-Rhinoceros-Annotation
@2807cc3 ships ONLY the final annotation as Git-LFS files
(GCA_021442165_annotation.gtf 225 MB, .gff3 154 MB) + README. No scripts.
The Galaxy workflow is a PDF supplement (S1 File). Raw RNA-seq: GEO GSE261038 (6
samples) + GSE300824 (8 samples).
In scope (pipeline-derived, attempted)
| Result | Pipeline | Reproducible? |
|---|---|---|
| Table 2: # transcripts (34,385) | StringTie2 merged assembly → count from shipped GTF | YES — count features in shipped GTF |
| Table 2: avg transcript length (36,981 bp) | derived from GTF coords | YES — compute from shipped GTF |
| Table 2: % genome covered (48%) | transcript span / genome size | YES — GTF + genome FASTA |
| Table 2: # genes (15,738) | StringTie2 + curation gene grouping | PARTIAL — file naming mixes SwissProt names + symbols |
| BUSCO completeness (93.4%) | BUSCO on transcriptome | YES (soft) — re-run BUSCO; lineage/version/mode unspecified in paper |
| Shipped-file integrity | git-lfs SHA256 | YES |
Out of scope (wet-lab / manual / unspecified — NOT attempted)
- Manual curation itself (BLAST-based, ≥80% identity, subjective gene-by-gene validation; the headline "+81% genes" claim depends on this). Not parameterized, not reproducible.
- HISAT2 read alignment of the 14 RNA-seq samples from raw FASTQ. Heavy (the hard 20%); and it would NOT reproduce the curated Table-2 numbers anyway (those come post-curation). Deliberately skipped per 80/20 — see AUDIT.md.
- Original-annotation correction counts (7,299 aligned / 6,763 / 212 / 455 bacterial genes removed): products of manual curation, not pipeline-derivable.
Approach
Reproduce by re-deriving the Table-2 pipeline statistics directly from the paper's own shipped annotation (P16: applying tools to the paper's data is valid), and by running BUSCO (third-party) on the annotation transcriptome. All compute on «our HPC»; data on «infra».
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.
Reproducing directly from the paper's own sha256-verified shipped annotation, 3 of 4 numeric Table-2 claims match within a few percent (transcripts 32,975 vs 34,385, length 37,615 vs 36,981 bp, coverage 49.7% vs 48%) once the curated symbol-bearing subset is isolated — no fabrication evidence, the reported values are consistent with the shared data. The main weaknesses are on the authors'/packaging side: the repo ships the full uncurated StringTie superset (475,980 tx) rather than the curated set, no scripts are provided (Galaxy workflow is a PDF), and the 15,738 gene count is not cleanly derivable due to mixed SwissProt/HGNC naming. BUSCO (93.4%) could not be tested due to a tool crash, and the headline +81%-genes manual-curation claim was out of scope, so the central conclusion is only partially confirmed. Overall a solid partial reproduction with explainable, mostly input/definition-level deviations — yellow.
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
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.