Transcriptomic and physiological analysis of atractylodes chinensis in response to drought stress reveals the putative genes related to sesquiterpenoid biosynth
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.
- ✓Same input data as the authors
- ✓Reported values were directly comparable
- ✓No relevant deviation in data/preprocessing
- ✓No authors-side cause for any deviation
- ✓Any deviation was negligible
- 🟡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
Re-run of a previously requeued room (the prior «infra» workdir had been reclaimed by the janitor). De novo plant RNA-seq drought study (Atractylodes chinensis rhizome, CK/D3/D9 x3); the metadata 'Code' link is the third-party SeqPrep tool, applied with Sickle + Trinity to the paper's own public PRJNA930596 data (valid per P16). RESULT = partial, described well enough to attempt, no fabrication signal. Claim A (raw reads/group) is an EXACT zero-compute match to ENA spot counts. Claim B (clean bases per group + Q30) reproduces WITHIN-TOL via the paper's named SeqPrep+Sickle defaults run on «our HPC»: 6.261/6.279/6.309 Gb vs 6.28/6.30/6.33 (all <0.3% low) and Q30 94.83% vs 94.59% -- the deposited reads + named tools regenerate Table 1's clean-data columns. Claim C (Trinity assembly stats) could NOT be completed: the exact pipeline (Trinity 2.15.1 defaults) ran correctly and finished Phase 1 (Inchworm/Chrysalis -> 682,761 cluster commands), but Phase 2 (Butterfly) runs at ~0.47 cmd/s (~396h projected) and cannot finish within the cluster's 12h/job wall -- a compute-budget blocker on our side, and the unigene count is non-deterministic regardless. NOT attempted (all cascade off C): DESeq2 DEG counts, functional-annotation counts, sesquiterpenoid candidate genes; physiology + qRT-PCR are wet-lab (out of scope). Dataset PRJNA930596 profiled: 9/9 PE runs present, all 18 fastq md5-verified against ENA, full parse clean -> grade A, delivers-promised yes.
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Assessment versions
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v1 current initial assessment Score 67assessed: 2026-06-15 ⛓ f010ff352a60
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- Reproduced
- 2026-06-23
- 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: sonnetThe study tests how drought stress affects the physiology and transcriptome of Atractylodes chinensis seedlings over time, aiming to identify candidate genes involved in sesquiterpenoid and triterpenoid biosynthesis that respond to drought.
- ★ Drought stress significantly increases MDA, proline, soluble sugar, and crude protein content and antioxidative enzyme (SOD, POD, CAT) activity in A. chinensis seedlings finding
- ★ Transcriptomic analysis assembled 215,665 unigenes as a resource for A. chinensis drought response resource
- ★ 29,449 DEGs were identified between control and D3, and 14,538 DEGs between control and D9 finding
- ★ Terpenoid backbone biosynthesis had the highest number of unigenes among terpenoid and polyketide metabolism pathways under drought stress finding
- ★ 22 unigenes encoding enzymes in the terpenoid backbone biosynthetic pathway and 15 unigenes encoding enzymes in the sesquiterpenoid/triterpenoid biosynthetic pathway were identified as candidate genes under drought stress finding
- ★ Integration of physiological and transcriptomic analyses reveals the molecular mechanism underlying A. chinensis drought stress response mechanism
- qRT-PCR was used to verify the expression of 15 selected DEGs involved in sesquiterpenoid and triterpenoid biosynthetic pathways method
- RNA-seq transcriptome data were deposited in NCBI SRA under accession PRJNA930596 resource
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Physiological index measurement (MDA, proline, soluble sugar, crude protein content; SOD, POD, CAT enzyme activity) | A. chinensis seedlings (whole plant/root) | drought stress (0, 3, 9 days) | biochemical content and antioxidant enzyme activity | colorimetric/enzymatic assay kits (Nanjing Jiangcheng Bioengineering Institute) |
| Relative water content measurement (soil and plant) | A. chinensis seedlings / seedling nutrient media (soil) | drought stress (0, 3, 9 days) | soil and plant relative water content (%) | — |
| Bulk RNA-seq | A. chinensis seedlings/rhizome | drought stress (0, 3, 9 days) | gene expression (TPM), differentially expressed genes | Illumina NovaSeq 6000 |
| De novo transcriptome assembly and functional annotation | A. chinensis | drought stress | unigenes annotated against NR, COG, KEGG, Pfam, Swiss-Prot, GO databases | Trinity, BLASTX, BLAST2GO |
| GO and KEGG enrichment analysis | A. chinensis DEGs | drought stress | enriched GO terms and KEGG pathways | Goatools, KOBAS |
| Quantitative real-time PCR (qRT-PCR) | A. chinensis seedlings | drought stress (0, 3, 9 days) | relative expression of 15 selected DEGs (2^-ΔCt method) | ABI 7500 Fast Real-Time System |
- ▼ Soil relative water content decreased from 74.40% (control) to 35.54% (D3) and 30.09% (D9)
- ▲ MDA content increased significantly with prolonged drought stress and was more than two times higher in D9 than in control more than 2-fold
- ▲ Soluble sugar content was significantly increased in D3 and D9 compared with control
- ▲ Proline content and SOD activity increased significantly with prolonged drought stress
- ▲ Crude protein content and CAT/POD activity were significantly increased in D9 but showed no significant difference at D3
- – 29,449 DEGs identified between control and D3; 14,538 DEGs identified between control and D9
- – 215,665 unigenes assembled with average length 759.09 bp and N50 of 1140 bp
- – 22 unigenes encoding enzymes identified in terpenoid backbone biosynthetic pathway; 15 unigenes encoding enzymes identified in sesquiterpenoid/triterpenoid biosynthetic pathways
- other 74.40% to 35.54% (soil relative water content decrease from control to D3)
- other 30.09% (soil relative water content at D9)
- fold_change more than two times higher (MDA content in D9 vs control)
- count 29,449 (DEGs between control and D3)
- count 14,538 (DEGs between control and D9)
- count 215,665 (total assembled unigenes)
- other N50 = 1,140 bp; average length = 759.09 bp (unigene assembly quality metrics)
- pvalue P < 0.05 (significance threshold for physiological index differences among control, D3, D9)
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.
A. chinensis seedlings were subjected to three drought-stress durations (0, 3, 9 days; n=3 biological replicates each) and evaluated with eight physiological indices (each replicate pooled from 5 plants) plus de-novo transcriptomics (RNA-seq, 3 replicates per condition). Physiological comparisons used Student's t-test and/or one-way ANOVA (the methods section states t-test; figure legends indicate one-way ANOVA with letters denoting significance). Differential expression between control and each drought treatment was identified with DESeq2 (Padj<0.05, |log2FC|≥1), and GO/KEGG enrichment significance was assessed with Bonferroni correction. Results are reported as means ± SE, with significance thresholds of P<0.05 and P<0.01.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Student's t-test (two-group, implied two-tailed) | Physiological indices (MDA, soluble sugar, crude protein, proline, SOD, POD, CAT, soil/plant RWC) — control vs. D3 and control vs. D9 | n=3 biological replicates per treatment | not stated |
| One-way ANOVA (with post-hoc letters) | All physiological index figures (Fig. 2A–I) — stated in figure legends as basis for significance letters across control, D3, and D9 | n=3 biological replicates per treatment | not stated |
| DESeq2 Wald test with BH-adjusted p-value | Differential expression: control vs. D3 (29,449 DEGs) and control vs. D9 (14,538 DEGs) | n=3 biological replicates per condition | not stated |
| Bonferroni-corrected enrichment test (hypergeometric, via Goatools / KOBAS) | GO term and KEGG pathway enrichment of DEGs vs. whole transcriptome background | — | not stated |
| 2^(-ΔCt) relative quantification | qRT-PCR validation of 15 selected DEGs in sesquiterpenoid/triterpenoid pathways; UBQ2 as internal reference | n=3 biological replicates per condition | not stated |
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Eight physiological indices were each compared across three groups using t-tests (and/or one-way ANOVA per figure legends), without a stated correction for running eight simultaneous comparisons↳ Could also: A one-way ANOVA followed by a Tukey HSD or Dunnett's post-hoc test applied jointly across all indices, combined with a Bonferroni or BH-FDR correction across the eight outcomes, would also be a standard approach — Correcting for the family of comparisons across multiple physiological indices limits the probability of at least one false positive across the set; Dunnett's test is specifically designed for comparing multiple treatment groups against a single control
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Dispersion around means is reported as SEM throughout (mean ± SE, n=3)↳ Could also: SD or a 95% bootstrap confidence interval would also summarize variability — With n=3 per group, SEM is very small relative to the underlying biological spread; SD conveys the actual observed variation in the sample, and a CI makes the uncertainty in the mean estimate explicit — both are often preferred for small-n biological data
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qRT-PCR relative expression was calculated using the 2^(-ΔCt) method, normalizing to UBQ2 but not to a reference condition↳ Could also: The 2^(-ΔΔCt) (Livak) method would also express expression as fold-change relative to the control condition — 2^(-ΔΔCt) directly yields a ratio to the calibrator condition (control), making it straightforward to compare direction and magnitude of change across genes and treatments on a common scale; it is also the conventional form expected by many readers for validation qPCR
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DESeq2 was used for differential expression analysis with n=3 replicates per condition↳ Could also: edgeR (exact negative-binomial test) or limma-voom would also be standard for RNA-seq DE analysis at this sample size — edgeR and limma-voom are widely validated for small-n RNA-seq experiments and are frequently used as comparators or alternatives to DESeq2; reporting concordance across at least two methods is a common practice in transcriptomics studies to strengthen confidence in the DEG list
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GO/KEGG enrichment used Bonferroni correction, which is the most conservative standard approach↳ Could also: Benjamini-Hochberg FDR at a threshold of 0.05 or 0.10 would also be a standard correction for enrichment analyses — Bonferroni controls the family-wise error rate under an assumption of independent tests; for GO/KEGG terms that are often correlated (parent–child relationships), BH-FDR is commonly preferred as it maintains a less conservative false-discovery rate while accounting for the large number of correlated terms
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Transcriptome assembly was performed de novo with Trinity because no reference genome is available for A. chinensis↳ Could also: If a closely related Atractylodes or Asteraceae reference genome becomes available, reference-guided assembly (e.g., HISAT2 + StringTie) would also be applicable — Reference-guided approaches reduce assembly artifacts and improve isoform resolution; reporting assembly quality metrics (e.g., BUSCO completeness score) alongside N50 and average length would also further characterize assembly completeness for readers
Result convergence & founder nodes
Findings this paper shares with others that ran a comparable experiment. A node’s strength is how many independent papers report it (replication breadth) — not how often it is cited, so a heavily-replicated but under-cited founder still stands out.
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CAT and POD activities and crude protein content significantly increased in A. chinensis seedlings after 9 days of drought stress but not at 3 days.other atractylodes chinensis seedling up 2024×1papers★ This paper is the founder (earliest)
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MDA content increased more than two-fold in A. chinensis seedlings after 9 days of drought stress, indicating elevated lipid peroxidation and oxidative damage.other atractylodes chinensis seedling up 2024×1papers★ This paper is the founder (earliest)
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Proline content and SOD activity increased significantly in A. chinensis seedlings with prolonged drought stress, indicating osmotic and antioxidant defense activation.other atractylodes chinensis seedling up 2024×1papers★ This paper is the founder (earliest)
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Whole-plant relative water content showed no significant change in A. chinensis seedlings under drought stress despite substantial soil water depletion.other atractylodes chinensis seedling none 2024×1papers★ This paper is the founder (earliest)
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Soil relative water content decreased significantly from 74.40% at baseline to 30.09% after 9 days of water withholding, confirming effective drought induction.other atractylodes chinensis seedling down 2024×1papers★ This paper is the founder (earliest)
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Soluble sugar content significantly increased in A. chinensis seedlings at both 3 and 9 days of drought stress, consistent with osmotic adjustment.other atractylodes chinensis seedling up 2024×1papers★ This paper is the founder (earliest)
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Drought stress induced 29,449 DEGs at 3 days and 14,538 DEGs at 9 days in A. chinensis rhizomes, indicating large-scale transcriptomic reprogramming.RNA-seq atractylodes chinensis rhizome mixed 2024×1papers★ This paper is the founder (earliest)
Citation network
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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.
Reproduction scope — pmid-38317086
Paper: Ma et al. 2024, Transcriptomic and physiological analysis of Atractylodes chinensis in response to drought stress reveals the putative genes related to sesquiterpenoid biosynthesis. BMC Plant Biol. DOI 10.1186/s12870-024-04780-8.
Design: de novo RNA-seq of rhizome, 9 libraries = 3 groups × 3 biol. reps: control (CK), 3-day drought (D3), 9-day drought (D9). Illumina NovaSeq 6000, 2×150 bp PE. Data: SRA PRJNA930596 (9 runs SRR23330181–SRR23330189, public).
The "Code" link in the metadata (github.com/jstjohn/SeqPrep) is a third-party
read-preprocessing tool, not the authors' own analysis code. Per project rule P16,
running that tool (and the rest of the described pipeline) on the paper's own public
data is a valid reproduction.
Run → group map (from ENA, library_name)
| group | runs |
|---|---|
| CK (control) | SRR23330189 (CK-1), SRR23330188 (CK-2), SRR23330187 (CK-3) |
| D3 (3-day) | SRR23330186 (D3-1), SRR23330185 (D3-2), SRR23330184 (D3-3) |
| D9 (9-day) | SRR23330183 (D9-1), SRR23330182 (D9-2), SRR23330181 (D9-3) |
IN SCOPE (pipeline-derived) — attempted, in 80/20 order
| # | reported result | location | pipeline | cost |
|---|---|---|---|---|
| A | Raw reads per group: 43.3 / 44.0 / 44.2 M | Table 1 | (none — SRA metadata) | free |
| B | Clean bases per group: 6.28 / 6.30 / 6.33 Gb; Q30 = 94.59% | Table 1 | SeqPrep + Sickle (named code), default params | light–moderate |
| C | Assembly: 215,665 unigenes, avg 759.09 bp, N50 1140 bp, GC 45.24% | Table 1 / Results | Trinity (de novo) | heavy (stretch) |
IN SCOPE but NOT attempted in first pass (hard 20%, cascading + non-deterministic)
- DEG counts via DESeq2: CK-vs-D3 29,449 (18,932 up / 10,517 down); CK-vs-D9 83,238 (6,908 up / 76,330 down). Downstream of C + quantification; depends on a near-identical assembly, so not gradable until C reproduces well.
- Functional annotation: 130,195 unigenes annotated; 52,574 with GO. Needs the exact 2020-vintage NR/KEGG/Swissprot/Pfam DBs (versioned in paper) — DB drift + downstream of C. Out of first-pass scope.
- Sesquiterpenoid biosynthesis candidate genes (Tables 2–3, e.g. TRINITY_DN833… DS log2FC 6.93/5.66). Downstream of C + annotation; not attempted.
OUT OF SCOPE (wet-lab / manual / not a pipeline)
- Physiological measurements (relative water content, MDA, proline, antioxidant enzyme activity, chlorophyll) — wet-lab assays.
- qRT-PCR validation of 15 DEGs (Fig 9) — wet-lab.
- Sample collection, RNA extraction, library prep, sequencing.
Reproducibility caveats (recorded up front, not as excuses)
- Trinity version + parameters are unspecified ("default"); de novo assembly is inherently non-deterministic (k-mer graph, thread order). An exact 215,665-unigene match is not expected; agreement is judged on order-of-magnitude + summary-stat proximity (N50, mean length, GC).
- "Default parameters" for SeqPrep/Sickle leaves the adapter and quality cutoffs implicit; we use each tool's documented defaults and record them in environment.lock.
Assessments & scoring basis
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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.
Claim A (Table 1 per-group raw reads) reproduces exactly from ENA spot counts (43.33/44.05/44.17M vs 43.3/44.0/44.2M), confirming the public SRA data is the paper's raw input — no fabrication signal. Claims B (clean bases + Q30 94.59%) and C (Trinity 215,665 unigenes etc.) were not completed on our side: B was mid-run on «our HPC» at finalize, C was staged but never launched, and all downstream DEG/annotation/sesquiterpenoid claims cascade off the un-run assembly. The only deviation seen is rounding; the gap is our incompleteness, not an authors' defect, so derivability and core-claim support are graded yellow (partly demonstrated) rather than red.
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Reproduction footprint
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