Expression and Secretion of Circular RNAs in the Parasitic Nematode, Ascaris suum.
Provisional — an automated or curator check raised a specific concern and points reviewers here. This is NOT a final assessment and not a determination about the authors.
The main result did not reproduce in this reproduction attempt. Where our recomputation produced values that differ from the published ones, those discrepancies are listed below. This is a single automated attempt — not peer review and not a finding of error or misconduct — and differences can also arise from data access, undocumented parameters or the computing environment. The verdict can be contested via “report an error”.
- ✓Reported values were directly comparable
- 🟡Could not use the authors’ exact input data
- 🟡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
MISMATCH. Authors' code (github.com/ISUgenomics/Kimber) is gone (404); per rule P16 we re-ran the DESCRIBED pipeline (cutadapt -> STAR-chimeric -> CIRCexplorer2, STAR substituting deprecated TopHat-Fusion) on the paper's own data (ENA PRJNA750737, 6 libs) against WBPS19 PRJNA62057. SLURM «job» completed (59m, exit 0). Reproduction is QUALITATIVELY PARTIAL but QUANTITATIVELY a mismatch: circRNAs are detected, one scaffold (AgR002) dominates ~40%, the exonic class is the majority, and the AgR002_g269/g270 host region is a top contributor - all directionally matching the paper. But every headline count is 2.5-5x lower (C1 424 vs 1982, C2 474 vs 1978, C3 788 vs 1997) and the paper's near-total cross-tissue sharing (1963/1997 ~98%) is NOT reproduced (we see only ~14% shared; most circRNAs are tissue-specific - the opposite structure). Most likely mechanism: STAR uniquely maps only 9-35% of reads to the WBPS19 scaffold genome (SRR15295819 only 9.35%, near-failed), so far fewer back-splice reads survive; compounded by the aligner/caller substitution and possible genome-build mismatch. The paper also has an internal inconsistency (C7 says AgR001 dominant, C8's top host gene AgR002_g270 is on AgR002). No fabrication evidence - the gap is consistent with method/genome substitution. The paper's description is INSUFFICIENT for exact reproduction (missing code, under-specified detection thresholds + cross-tissue union rule, ambiguous assembly version). Datasets profiled: PRJNA750737 grade A (complete, balanced; instrument-model metadata blemish HiSeq4000 vs paper's NovaSeq6000), PRJNA62057 grade B (open, usable, but AGO1 version ambiguity blocks ID-level reproduction). NOT attempted: all wet-lab/manual claims (out of scope).
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 50assessed: 2026-06-19 ⛓ cd027c6e65e6
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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
- 2026-06-26
- 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: sonnetThe authors hypothesized that Ascaris suum expresses endogenous circular RNAs (circRNAs) that may function as miRNA sponges, and that a subset of these circRNAs would be secreted in extracellular vesicles (EVs) and interact with host miRNAs at the host-parasite interface.
- ★ A. suum expresses 1,997 distinct circRNAs identified via next-generation sequencing in adult female body wall and ovary-enriched tissue finding
- ★ circRNA expression is broadly shared between body wall and ovary-enriched tissue, with only 34 circRNAs showing tissue-specific expression finding
- ★ circRNAs are present in EVs secreted by A. suum into the external environment, the first such report in a parasitic helminth finding
- ★ In-silico analysis predicts that a subset of Ascaris circRNAs bind both endogenous Ascaris miRNAs and human host miRNAs finding
- ★ Human miRNAs are predicted to form more thermodynamically stable interactions with Ascaris circRNAs than Ascaris miRNAs do, with no strong correlation between circRNA length and endogenous miRNA binding finding
- ★ Ivermectin treatment does not directly alter Ascaris circRNA expression or secretion, despite previously inhibiting EV release finding
- Dual de novo circRNA assembly pipeline (CIRCExplorer with Bowtie2/Tophat2 and Tophat-fusion mapped reads) used to identify circRNAs from NGS data method
- qRT-PCR with divergent primers targeting back-splice junctions used to validate circRNA expression in tissue and EV samples method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| circRNA sequencing (RNase R-treated RNA, PE150 Illumina) | A. suum adult female body wall muscle tissue | none | circRNA identification and expression | Illumina Novaseq 6000 |
| circRNA sequencing (RNase R-treated RNA, PE150 Illumina) | A. suum adult female ovary-enriched tissue | none | circRNA identification and expression | Illumina Novaseq 6000 |
| circRNA sequencing | A. suum secreted extracellular vesicles (from conditioned media) | none | circRNA presence in EV cargo | Illumina Novaseq 6000 |
| qRT-PCR (back-splice junction divergent primers, exogenous spike-in standard) | A. suum body wall/ovary tissue and EV/EV-depleted media samples | none | circRNA abundance validation | Power Up SYBR Green Master Mix |
| drug treatment with qRT-PCR readout | A. suum adult worms (body wall/ovary tissue and conditioned media) | ivermectin, diethylcarbamazine, or levamisole (0.1 or 1 µM) vs 0.1% DMSO vehicle | circRNA expression and secretion fold change (2^-ΔΔCq) | — |
| EV isolation and quantification (differential ultracentrifugation, NTA, TEM) | A. suum conditioned media / secreted EVs | none | EV size and concentration | NanoSight LM10 (NTA); JEOL 2100 TEM |
| in-silico miRNA target prediction (miRanda, TargetScan) | A. suum circRNA sequences vs. A. suum and human miRNA datasets (miRBase) | none | predicted circRNA-miRNA binding sites, free energy/score | miRanda; TargetScan |
- – 1,997 distinct circRNAs identified in A. suum (1,982 in body wall, 1,978 in ovary-enriched tissue) 1,997 total
- – 1,963 circRNAs shared between tissues; only 34 tissue-specific (15 ovary-specific, 19 body wall-specific) 34/1997
- – circRNAs (exonic, intronic, intergenic) detected in EVs secreted by Ascaris
- – Subset of Ascaris circRNAs predicted to bind both Ascaris and human miRNAs
- – No strong correlation between circRNA length and number of endogenous miRNA interactions
- ▲ Human miRNAs predicted to form more thermodynamically stable bonds with Ascaris circRNAs than Ascaris miRNAs
- – Ivermectin treatment did not change circRNA expression or secretion relative to vehicle control
- count 1,997 (total unique circRNAs identified across A. suum body wall and ovary-enriched tissue)
- count 1,982 (circRNAs identified in body wall tissue)
- count 1,978 (circRNAs identified in ovary-enriched tissue)
- count 1,963 (circRNAs shared between body wall and ovary-enriched tissue)
- count 34 (15 ovary-specific, 19 body wall-specific) (tissue-specific circRNAs)
- pvalue p < 0.05 (significance threshold for two-way ANOVA comparing circRNA concentrations between treatments)
- other miRanda free energy 200-140; TargetScan score -0.13-0 (thresholds used to assign circRNA-miRNA interaction confidence)
- count 10 (sets of divergent primers designed for qRT-PCR validation of circRNA back-splice junctions)
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.
The study is a descriptive/exploratory sequencing study of circular RNAs in Ascaris suum tissues and extracellular vesicles, combined with a separate drug-treatment experiment. Differential circRNA expression between tissue/mapping comparisons was assessed with DESeq2, followed by GO/KEGG enrichment analysis; qRT-PCR (2^-ΔΔCq, log2-transformed) was used for validation and drug-treatment quantification. Effects of anthelmintic drug treatment on circRNA concentration were tested with a two-way ANOVA with multiple comparisons in GraphPad Prism, using p<0.05 as the significance threshold, with each 'N' defined as a biologically distinct batch of worms with its own control.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| DESeq2 (default Wald-test-based differential expression) | Differential circRNA expression between body wall and ovary-enriched tissue samples, analyzed separately for unique and multiple mapping reads | Three ovary-enriched and three body wall biological samples | not stated |
| GO and KEGG enrichment analysis (clusterProfiler, Ontologizer) | Enrichment analysis of differentially expressed circRNAs | — | not stated |
| Two-way ANOVA with multiple comparisons (GraphPad Prism) | Comparison of circRNA concentrations (qRT-PCR-derived) between drug treatments (ivermectin, diethylcarbamazine, levamisole at two concentrations vs. vehicle control) and sample types | Each N = one biologically distinct batch of worms with its own no-treatment control; exact number of batches not stated in the excerpt | not stated |
| Nonlinear (second-degree polynomial) least-squares regression | Standard curve fit for the exogenous spike-in RNA used to quantify circRNA abundance by qRT-PCR | — | not stated |
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Differential circRNA expression was assessed using DESeq2 on unique and multiple mapping reads in separate comparisons.↳ Could also: edgeR or limma-voom — These are also widely used RNA-seq differential expression frameworks with different dispersion estimation approaches, and comparing results across methods can help characterize the robustness of detected expression differences, particularly with small sample sizes.
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The two-way ANOVA for drug-treatment circRNA concentrations used a 'multiple comparisons' procedure without naming the specific post-hoc correction.↳ Could also: Explicitly specifying and reporting a named method such as Tukey's HSD, Sidak's correction, or a Benjamini-Hochberg FDR adjustment — Naming the specific correction lets readers evaluate exactly how the family-wise error rate or false discovery rate was controlled across the multiple treatment/dose comparisons.
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Significance was reported using a p<0.05 threshold without exact p-values.↳ Could also: Reporting exact p-values alongside the threshold — Exact p-values (and effect sizes) allow readers to gauge the strength of evidence more precisely rather than only whether a fixed cutoff was crossed.
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Drug-treatment comparisons relied on a parametric two-way ANOVA.↳ Could also: A nonparametric alternative such as the Kruskal-Wallis test with post-hoc comparisons — With small biological batch sizes, a nonparametric approach can be preferred when normality of the underlying data is uncertain and does not need to be assumed.
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Variability in circRNA concentration/expression data is not described with a stated dispersion measure in the excerpted methods.↳ Could also: Reporting SD, SEM, or 95% confidence intervals alongside means — Explicitly stating a dispersion measure communicates the spread and precision of the estimates directly to readers.
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qRT-PCR quantification relied on a single exogenous spike-in RNA standard curve for extrapolating circRNA concentration.↳ Could also: Normalization using multiple reference transcripts or endogenous control genes — Using several reference points can help cross-validate relative quantification and account for variability introduced by a single spike-in standard.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-35711944
Paper: Minkler SJ, Loghry-Jansen HJ, Sondjaja NA, Kimber MJ (2022). "Expression and Secretion of Circular RNAs in the Parasitic Nematode, Ascaris suum." Front Genet 13:884052. DOI 10.3389/fgene.2022.884052. PMID 35711944 / PMC9194832.
Data the paper relies on
- RNA-seq (the data WE reproduce on): BioProject PRJNA750737, runs
SRR15295818–SRR15295823 (6 libraries: 3 ovary-enriched, 3 body-wall muscle;
Illumina NovaSeq 6000, 2×150 bp PE). Confirmed open on ENA.
NOTE: the room BRIEF listed
PRJNA62057as the data accession — that is actually the reference genome BioProject (see below), not the RNA-seq. Corrected here. - Reference genome: A. suum BioProject PRJNA62057, the paper says assembly "AGO1" (Wang J. et al. 2017, chromosome-level germline assembly; chromosomes named AgR0xx / AgB0xx). WormBase ParaSite hosts PRJNA62057 as GCA_000187025.3 (ASM18702v3, scaffold-level). Whether the paper used the chromosome-level "AGO1" build or the WBPS scaffold build is a reproduction-relevant uncertainty (affects back-splice junction coordinates and the AgR/AgB chromosome IDs quoted).
Code availability
- Paper cites
https://github.com/ISUgenomics/Kimberfor "notes and scripts used to produce expression analysis." This repo is GONE (HTTP 404; the ISUgenomics org exists with 63 repos, none named Kimber; no archived snapshot found at first try). - Per reproduction rule P16, applying the described third-party pipeline (CIRCexplorer + TopHat-Fusion) to the paper's own data is equally valid. We reproduce by re-running the described pipeline, not the (missing) author scripts.
Pipeline described in Methods
- QC/trim: Cutadapt, FastQC
- circRNA detection: CIRCexplorer ("dual de novo assemblies of circular RNAs"), using Bowtie2 / TopHat2 / TopHat-Fusion alignment of the back-spliced reads.
- Expression quant: HISAT2 v2.2.0, Samtools v1.10, featureCounts (Subread v1.6.0).
- Differential expression: DESeq2 v1.20.0.
- Enrichment: clusterProfiler, Ontologizer.
- circRNA–miRNA interaction prediction: (miRanda/TargetScan-class; tool/version not fully specified in abstract-level read — to confirm from full Methods).
IN SCOPE (pipeline-derived, attempted)
| id | reported result | pipeline |
|---|---|---|
| C1 | 1,982 circRNAs in body wall | CIRCexplorer de novo on body-wall libs |
| C2 | 1,978 circRNAs in ovary | CIRCexplorer de novo on ovary libs |
| C3 | 1,997 total distinct circRNAs (union) | union of C1+C2 |
| C4 | 1,963 shared between tissues | intersection |
| C5 | 15 ovary-specific / 19 body-wall-specific | set difference |
| C6 | composition: 1,178 exonic (59%) / 779 intronic (39%) / 40 intergenic (2%) | CIRCexplorer annotation vs GFF |
| C7 | 752 (37%) circRNAs from chromosome AgR001 | per-chromosome tally |
| C8 | AgR002_g270 host gene → 303 circRNAs (15%) | per-gene tally |
OUT OF SCOPE (wet-lab / manual / not pipeline-reproducible)
- EV isolation, NTA mean EV size 194 nm (instrument measurement).
- RT-PCR validation that 2 circRNAs (AgR007_g109_t01, AgB08X_g209_t01) are EV-secreted.
- Drug-treatment (ivermectin/DEC/levamisole) wet-lab handling.
- miRNA-interaction biological interpretation (the counts 202/398 are pipeline-derived → secondary in-scope target C9/C10 if the miRNA inputs + tool are recoverable).
Reproduction strategy
- Front1: download genome (resolve AGO1 vs GCA_000187025.3) + 6 SRA fastqs to «infra».
- SLURM: Cutadapt trim → TopHat2/TopHat-Fusion → CIRCexplorer de novo per library.
- Count distinct back-splice junctions per tissue; union/intersection; annotate composition + per-chromosome/per-gene tallies. Compare to C1–C8.
- Honest 1:1; document assembly/version mismatches as provisional.
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.
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.