Organelle Genomes and Transcriptomes of Nymphaea Reveal the Interplay between Intron Splicing and RNA Editing.
The main results reproduced: recomputed values matched the published ones within tolerance.
Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
- ✓Any deviation was negligible
- 🟡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 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
PARTIAL reproduction. The deposited organelle genomes (GenBank MW644616/MW644617) reproduce the paper's headline structural claims essentially 1:1: chloroplast 159,968 bp and mitochondrion 335,042 bp are BIT-EXACT to the reported sizes, and gene contents match within +/-1 gene (cp 80 PCG/4 rRNA exact, 30->31 tRNA; mt 3 rRNA/21 tRNA exact, 41->40 PCG) with discrepancies fully explained by annotation-counting convention (unique gene name vs feature copy, IR duplicates, a doubly-annotated trnG). The named SRA run SRR15402840 was downloaded and profiled: 1667 subreads / 3,907,546 bases (EXACT match to ENA metadata), 97.1% of reads map to the deposited organelle genomes (214 cp + 1405 mt), confirming it is genuine Nymphaea organelle Iso-Seq data. NOT ATTEMPTED / NOT REPRODUCIBLE: the Iso-Seq aggregate numbers (441,656 corrected full-length transcripts; 3121/4190 mapped to cp/mt) and the RNA-editing-site counts (98 cp, 865 mt) and the cis/trans intron tallies require (a) all 8 SRA runs SRR15402840-847 plus 74 Gb Illumina (this RU names only run ...840), and (b) the proprietary SMRTLINK 5.0.1 Iso-Seq v2 / ICE-Arrow / LoRDEC / GMAP pipeline described in Methods. NOTE: the repo listed for this RU (cDNA_Cupcake, github.com/Magdoll) is NOT used in the paper's Methods at all -- it is a likely text-mining false-positive; the paper's pipeline is SMRTLINK + LoRDEC + GMAP. Verdict is provisional and must be human-audited.
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Assessment versions
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v1 current initial assessment Score 76assessed: 2026-06-18 ⛓ 3ab56b1fab54
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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
- 2026-06-18
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-19no 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: opusHow do intron-splicing intermediates accumulate and interact with RNA editing in plant organelle genomes, and can long-read transcriptomics capture the full spectrum of splicing intermediates and their interplay with RNA editing in the basal angiosperm Nymphaea?
- ★ Multiple partially or fully intron-spliced intermediates co-exist within an organelle, and both cis- and trans-splicing introns are spliced randomly (no fixed order), generating diverse intermediates. finding
- ★ 98 and 865 C-to-U RNA-editing sites were identified in the plastome and mitogenome of N. 'Joey Tomocik', respectively. finding
- ★ RNA-editing sites in intron and exon regions may splice synchronously, except exonic sites adjacent to introns which can only be edited after intron splicing. mechanism
- ★ Shared target codon preference, increased protein hydrophobicity, and biased editing-site distribution in both organelles suggest a common evolutionary origin and shared editing machinery. finding
- ★ Combining PacBio DNA-seq/Iso-seq long reads with Illumina short reads and rRNA-depleted strand-specific RNA-seq, plus direct SNP-calling and transcript-mapping, captures full transcript intermediates and editing landscape. method
- ★ Complete chloroplast (159,968 bp) and mitochondrial (335,042 bp) genomes of N. 'Joey Tomocik' were assembled and annotated. resource
- Full-length Iso-seq transcripts corrected misannotations (e.g., nad4 exon2/exon3 boundary, rpl16 exon2, cox2 exons) and detected co-transcribed polycistronic transcriptional units. finding
- Ancestral angiosperms possessed a relatively high level of organellar RNA editing with extensive independent loss across lineages; edited sites show no clear phylogenetic signal beyond total-number variation. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| PacBio single-molecule long-read DNA-seq (genome assembly) | Nymphaea 'Joey Tomocik' organelles | none | chloroplast and mitochondrial genome assemblies | PacBio (Organelle_PBA, Arrow, Pilon polishing) |
| Illumina short-read DNA-seq (genome assembly/cross-check) | Nymphaea 'Joey Tomocik' organelles | none | chloroplast (159,968 bp) and mitochondrial (335,042 bp) genome assemblies | Illumina (NOVOPlasty) |
| PacBio Iso-seq full-length transcript sequencing | Nymphaea 'Joey Tomocik' chloroplast and mitochondria | none | full-length transcripts; intron-splicing intermediates, co-transcribed PTUs, RNA-editing sites | PacBio Iso-seq (GMAP mapping) |
| Strand-specific RNA-seq (rRNA-depleted, non-Oligo(dT)) | Nymphaea 'Joey Tomocik' chloroplast and mitochondria | none | RNA-editing sites and editing efficiency (VAF) | Illumina ssRNA-seq; bcftools SNP-calling |
| Illumina RNA-seq de novo assembly (Trinity) | Nymphaea 'Joey Tomocik' organelles | none | transcript-mapping support for splicing events and editing sites | Trinity |
| In silico RNA-editing prediction | Nymphaea / Liriodendron organelle genomes | none | predicted C-to-U editing sites compared to experimental sites | PREPACT3 webserver |
| Comparative RNA-editing analysis from public sequence data | Liriodendron tulipifera and Amborella trichopoda plastomes/mitogenomes | none | RNA-editing site counts and shared/exclusive sites across basal angiosperms | — |
- – Identified RNA-editing sites in plastome and mitogenome of N. 'Joey Tomocik' 98 plastid; 865 mitochondrial
- – All eight possible intron-splicing intermediates detected in mitochondrial nad4 gene, supporting random (non-ordered) splicing 8 intermediates
- – Pyrimidine dominates the -1 position (5'-adjacent to edited C) in both organelle genomes >93%
- – Most editing sites efficiently edited (VAF >0.6) in both organelles >80% of sites
- – Non-synonymous editing sites: 89 plastid and 734 mitochondrial 89; 734
- – Splicing events detected: 25 by Iso-seq, 14 additional by Trinity 53.33% (Iso-seq); 86.67% (combined)
- – PREPACT3-predicted sites consistent with experimental editing sites 76 plastid, 615 mitochondrial (>70% of total)
- – Plastid plastome RNA-editing sites in compared basal angiosperms 89 (L. tulipifera); 169 (A. trichopoda)
- count 98 and 865 RNA-editing sites (plastome and mitogenome) (N. 'Joey Tomocik' editing sites)
- count chloroplast genome 159,968 bp (final NOVOPlasty assembly)
- count mitochondrial genome 335,042 bp (final mitogenome assembly)
- count PacBio: 3,239,192 subreads (19.68 Gb), avg 6077 bp, N50 9200 bp (PacBio DNA-seq output)
- count Illumina DNA-seq 418,782,912 raw paired-end reads (73.92 Gb); 60.72 Gb clean (Illumina sequencing output)
- count 94 plastome and 807 mitogenome editing sites in coding regions (distribution; 36 plastid genes, all 41 mito genes)
- mean mitogenome avg editing efficiency: CDS 0.802, intron 0.796, UTR 0.693, intergenic 0.625 (VAF averages by region)
- other >86% editing efficiency for stop-codon-generating edits (petD; atp6, ccmFC) (RNA-editing-generated stop codons)
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 study is primarily a descriptive comparative genomics paper that assembled and characterized the chloroplast and mitochondrial genomes of a single Nymphaea 'Joey Tomocik' individual using PacBio long reads, Illumina short reads, Iso-seq, and strand-specific RNA-seq. Posttranscriptional features—intron splicing intermediates and RNA editing sites—were catalogued through bioinformatics pipelines (bcftools SNP-calling combined with transcript-mapping). Results were reported as counts, proportions, and mean variant allele frequencies (VAFs), with cross-species comparisons presented descriptively rather than through formal statistical hypothesis tests.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Variant allele frequency (VAF) calculation for RNA editing efficiency | Characterization of editing efficiency across plastome and mitogenome regions (Figure 3b); means reported per genomic region (CDS, intron, UTR, intergenic) | 98 plastome and 865 mitogenome editing sites | not stated |
| SNP calling (bcftools) | Identification of C-to-U RNA editing sites in organelle transcriptomes | — | not stated |
| Descriptive nucleotide frequency count | Pyrimidine dominance at the −1 position adjacent to edited cytidines (Figure 3d) | 98 plastome and 865 mitogenome editing sites | not stated |
| BLASTN sequence similarity search | Contig selection and mitogenome assembly validation against Nymphaea colorata reference | — | na |
| Prediction tool overlap (PREPACT3/REPACT3) | Comparison of predicted vs. experimentally identified RNA editing sites in plastome and mitogenome | 98 plastome; 865 mitogenome editing sites | not stated |
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Editing efficiency (VAF) values were summarized as means per genomic region (CDS, intron, UTR, intergenic) with no measure of spread↳ Could also: Report standard deviation, interquartile range, or 95% bootstrap confidence interval alongside each mean VAF — Measures of dispersion would convey the variability of editing efficiency across sites within each region, making group-level averages more interpretable and enabling comparison across regions
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Cross-species comparison of RNA editing site counts, codon position preference, and gene-level distributions was conducted descriptively↳ Could also: Apply chi-square or Fisher's exact tests to compare proportions of edited codons or positional biases between species pairs — Formal tests would quantify whether observed differences in editing frequency or codon-position preference exceed what is expected under sampling variation, providing a probability-based basis for comparative conclusions
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Nucleotide frequency at the −1 position adjacent to edited cytidines was described as 'pyrimidines dominating more than 93%' without a formal test↳ Could also: Apply a binomial test or chi-square goodness-of-fit test against a uniform or genomic-background nucleotide frequency null — A formal test would provide a probability statement about the magnitude of the observed pyrimidine enrichment relative to background, standardizing the comparison across organelles and species
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Phylogenetic signal in RNA editing sites was assessed by visual inspection of aligned edited positions across three basal angiosperm species↳ Could also: Quantify phylogenetic signal using Blomberg's K statistic or Pagel's lambda in a phylogenetic comparative framework — Standardized phylogenetic signal statistics provide a testable, quantitative measure of how strongly editing-site presence/absence covaries with the phylogeny, complementing pattern description
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Prediction tool performance (PREPACT3/REPACT3) was reported as raw overlap counts and percentages of experimental sites recovered↳ Could also: Report sensitivity, positive predictive value (precision), and F1-score or Cohen's kappa relative to the experimental dataset — Standard classification performance metrics give a more complete and symmetric picture of agreement between predicted and experimental editing sites than one-directional overlap counts alone
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The entire analysis is based on one individual, so all observed editing site counts and efficiencies are point estimates without within-species variance↳ Could also: Include multiple biological replicates (additional individuals) analyzed with replicate-aware quantification — Biological replication would allow estimation of within-species variability in editing site number and efficiency, enabling formal inference about whether patterns are species-level rather than individual-level observations
What was reproduced
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Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
The deposited organelle genomes reproduce the paper's headline structural claims essentially 1:1 — chloroplast 159,968 bp and mitochondrion 335,042 bp are bit-exact, and gene contents match within ±1, with every discrepancy explained by annotation-counting convention (unique name vs IR-duplicated feature copy, doubly-annotated trnG). The deviations are negligible and on the definition/our-method side, not the authors'. However, the paper's actual thesis (intron-splicing ↔ RNA-editing interplay: 98 cp + 865 mt editing sites, 441,656 transcripts) is untestable here because 7 of 8 SRA runs and the proprietary SMRTLINK 5.0.1 pipeline are unavailable — a data/software-availability gap, not fabrication. Net: a solid but partial reproduction; structural claims confirmed, transcriptomic core only limitedly verified.
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