Comprehensive transcriptome study to develop molecular resources of the copepod Calanus sinicus for their potential ecological applications.
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 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 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
Full pipeline (SRA download -> SeqPrep/Sickle QC -> Trinity 2.3.2 assembly -> TrinityStats -> MISA SSR mining) completed end-to-end on the HPC cluster via SLURM checkpoint-resume after the original 12h Trinity job (2443003) timed out at 12/12h wall time; resume «job» completed in 3m43s since all Butterfly partition commands had already finished, only the final Trinity.fasta collation remained. Input data fidelity is excellent: raw read count (58,944,478) matches the paper exactly, and QC retention (98.53% incl. singles) is close to the papers 98.0%. However the resulting assembly is far more fragmented than reported: 217,399 raw transcripts / 160,054 genes vs. the papers 69,751 post-redundancy-removal contigs, with N50 (483bp) at only 43% and average length (444bp) at only 48% of the papers values (1,127bp / 928.8bp) - most likely because the paper applied a redundancy-removal/clustering step after Trinity that this pipeline did not perform, and/or used a different Trinity version (unspecified in the paper beyond default settings, k-mer=25). SSR mining recovers 4,222 total SSRs via MISA default parameters, 87% of the papers reported 4,871 (Table 4) - a reasonable order-of-magnitude match despite the more fragmented input - and correctly reproduces the qualitative finding that trinucleotide repeats dominate (76.4% here vs 92.4% reported), though the exact type distribution (notably a higher dinucleotide share) does not match precisely.
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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
- 2026-08-02
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-08-02no human curator yet
- Last updated
- 2026-08-02
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 Illumina-based RNA-Seq of adult Calanus sinicus rapidly generate a comprehensive transcriptome and molecular-marker resource (microsatellites, SNPs, stress- and lipid-metabolism genes) for this non-model, ecologically dominant copepod? The study tests whether such a de novo approach yields validated markers usable for future population genetics and ecological applications.
- ★ Illumina RNA-Seq with Trinity de novo assembly produced a C. sinicus transcriptome of 69,751 contigs (average 928.8 bp, N50 1,127 bp) from 58.9 million reads. resource
- ★ Gene annotation against the NCBI nr database identified 43,417 unique protein hits, with GO, COG, and KEGG functional categorization. resource
- ★ 4,871 microsatellites and 110,137 putative SNPs were identified in the transcriptome sequences. resource
- ★ SNP validation by the melting temperature (Tm)-shift method showed 16 primer pairs amplified target products and displayed biallelic polymorphism among 30 individuals. finding
- ★ Transcripts potentially involved in stress response were identified, including heat shock proteins (HSP90, HSP70, HSP60, HSP40, HSP10), cytochrome P450s, glutathione S-transferases, and superoxide dismutases, proposed as candidate environmental biomarkers. finding
- ★ Genes involved in fatty acid/lipid metabolism (e.g., ELOV elongases, fatty acid binding proteins) were identified and are proposed to function in wax ester synthesis, transport, and storage in preparation for diapause. mechanism
- The Tm-shift genotyping method, using one common reverse primer plus two allele-specific forward primers bearing GC tails of 14 and 6 bases, discriminates SNP alleles by melting-curve shift. method
- Illumina-based RNA-Seq demonstrates power for rapid development of molecular resources in non-model species and serves as a guide for related copepod taxa. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| RNA-Seq (transcriptome sequencing, 100 bp paired-end) | Calanus sinicus adults, pool of ~50 individuals collected from the Yellow Sea (38°45′N, 121°45′E), May 2013 | none | short sequence reads / transcript abundance and sequence content | Illumina HiSeq 2000; TruSeq RNA sample preparation kit; RNeasy Mini Kit (Qiagen); NanoDrop (Thermo Scientific); Agilent 2100 Bioanalyzer with RNA 6000 Pico LabChip; Qiagen PCR extraction kit; KAPA quantitation |
| De novo transcriptome assembly (bioinformatic) | C. sinicus cleaned Illumina reads | none | contig number, length distribution, N50, average length | Trinity (fixed k-mer size 25); SeqPrep and Sickle for trimming |
| Sequence homology annotation (BLASTP/BLASTX) | C. sinicus Trinity contigs / predicted ORF protein sequences | none | number of significant hits (E-value ≤1e-5) and unique proteins | NCBI nr protein database, STRING database, KEGG GENES database |
| Gene ontology annotation | C. sinicus annotated unigenes | none | GO terms assigned per level-2 category (biological process, cellular component, molecular function) | Blast2GO 2.5.0 |
| KEGG pathway and COG functional classification | C. sinicus annotated unigenes | none | EC numbers assigned, number of pathways, percentage of sequences per functional group | KEGG; COG |
| Microsatellite (SSR) mining | C. sinicus transcriptome contigs | none | number and repeat-motif class of microsatellites | Msatcommander (criteria: 7 repeats dinucleotide; 5 for tri-, tetra-, pentanucleotide; 4 hexanucleotide) |
| SNP discovery by read alignment to reference transcriptome | C. sinicus Trinity-assembled transcripts as reference | none | number of putative SNPs, transition/transversion types, SNP frequency per kb, codon position | Samtools (minimum variant count 2 HQ bases; minimum site depth 8 HQ bases) |
| SNP genotyping by melting temperature (Tm)-shift allele-specific PCR | Genomic DNA from 30 wild individual C. sinicus from the Northern Yellow Sea | none | amplification success and biallelic polymorphism; observed and expected heterozygosity (Ho, He) | ABI 7500 real-time thermal cycler; SYBR Premix Ex Taq (Takara); Foregene genomic DNA isolation kit; POPGENE 32 |
- – 58.9 million 100 bp paired-end reads were generated, of which 57.7 million (98.0%) passed quality filtering; raw data deposited at NCBI SRA (SRP032493). 57.7 million of 58.9 million (98.0%)
- – Trinity assembly yielded 69,751 contigs with N50 of 1,127 bp and average length 928.8 bp; 45.3% were <600 bp, 34.0% were 600–1,200 bp, and 20.8% were >1,200 bp. 69,751 contigs; N50 1,127 bp; mean 928.8 bp
- – 58,885 assembled contigs had significant hits (E-value ≤1e-5) against the nr protein database, representing 43,417 unique proteins. 58,885 contigs / 43,417 unique proteins
- – 60 GO terms were assigned to 13,639 unigenes (23 biological process [38.3%], 19 cellular component [31.7%], 18 molecular function [30.0%]); 1,934 unigenes were annotated to response to stimulus (GO:0050896). 13,639 unigenes; 1,934 stimulus-response unigenes
- – EC numbers were assigned to 14,553 unigenes involved in 324 pathways; 45.5% fell into genetic information processing, 42.8% metabolism, 18.3% cellular processes, and 15.2% environmental information processing. 14,553 unigenes; 324 pathways
- – 4,871 microsatellites were identified, mostly trinucleotide (92.4%) and dinucleotide (4.8%) repeats, with AGG the predominant trinucleotide motif at 20.7% frequency. 4,871 SSRs; trinucleotide 92.4%
- – 110,137 putative SNPs were found, comprising 71,213 transitions (C/T 37,017; A/G 34,196) and 38,924 transversions (A/T 13,346; A/C 8,904; T/G 8,619; C/G 8,055); C/T was most common (33.6%) and C/G least common (7.3%); SNP frequency was 3.01 per kb and 83,270 (75.6%) occurred at the third codon position. 110,137 SNPs; 3.01 SNPs per 1 kb
- – Of 51 putative SNPs tested with Tm-shift primers, 16 (31.4%) were successful and showed biallelic polymorphism, 6 (11.8%) did not amplify any product, and 29 (56.9%) failed due to amplification failure of one allele-specific primer. 16/51 (31.4%) successful
- count 58.9 million 100 bp paired-end reads generated; 57.7 million high-quality sequences (98.0%) retained (Illumina HiSeq 2000 sequencing output and quality filtering)
- count 69,751 contigs; N50 = 1,127 bp; average length 928.8 bp (Trinity de novo assembly of the C. sinicus transcriptome)
- count 58,885 contigs with significant hits; 43,417 unique proteins (BLAST search against NCBI nr protein database)
- pvalue E-value cutoff 1e-5 (Significance threshold for BLASTP/BLASTX homology annotation)
- count 60 GO terms assigned to 13,639 unigenes; 1,934 unigenes in response to stimulus (GO:0050896) (Blast2GO level-2 functional categorization)
- count 14,553 unigenes assigned EC numbers across 324 pathways (KEGG pathway analysis of annotated unigenes)
- count 4,871 microsatellites; trinucleotide 92.4%, dinucleotide 4.8%; AGG motif frequency 20.7% (Msatcommander microsatellite mining)
- count 110,137 putative SNPs (71,213 transitions, 38,924 transversions); frequency 3.01 per 1 kb; 83,270 (75.6%) at third codon position; 16 of 51 (31.4%) validated as biallelic polymorphic (SNP discovery via Samtools and Tm-shift validation across 30 wild individuals)
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 primarily a descriptive genomic-resource paper rather than a hypothesis-testing study: it reports de novo transcriptome assembly (Trinity) and annotation (BLAST, Blast2GO, KEGG/COG mapping) of pooled Calanus sinicus RNA-Seq reads, followed by in silico identification of microsatellites and SNPs and empirical validation of a subset of SNPs by Tm-shift genotyping in 30 individuals. Quantitative results are reported mainly as counts, percentages, and rates (e.g., % GO categories, SNPs per kb, primer success rate), with observed/expected heterozygosity calculated in POPGENE 32 for the validated SNP loci. No inferential statistical hypothesis tests (e.g., t-tests, ANOVA, chi-square) are described in the text.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Observed and expected heterozygosity (Ho, He) calculation via POPGENE 32 | SNP polymorphism analysis of 16 validated loci across 30 wild individuals | 30 individuals | not stated |
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Observed and expected heterozygosities were calculated in POPGENE 32 for the 16 validated SNP loci, without a stated formal test of deviation from Hardy-Weinberg equilibrium.↳ Could also: An exact test for Hardy-Weinberg equilibrium (e.g., as implemented in GENEPOP or Arlequin) or a chi-square goodness-of-fit test — This would provide a formal p-value/probability for whether observed genotype frequencies differ from Hardy-Weinberg expectations at each locus, complementing the descriptive Ho/He values already reported.
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SNPs were called from aligned reads using fixed thresholds (minimum variant count of 2 high-quality bases and minimum site depth of 8) via Samtools.↳ Could also: A probabilistic/Bayesian variant caller (e.g., GATK HaplotypeCaller, FreeBayes, or bcftools with genotype-likelihood models) — Such tools output per-variant quality scores or posterior probabilities, which can convey calling confidence in addition to the count/depth thresholds used here.
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Transcriptome sequencing was based on RNA pooled from a single collection of ~50 individuals, without additional independent biological replicate pools.↳ Could also: Sequencing multiple independent biological replicate pools — Replicate pools would allow estimation of biological variance and, if expression comparisons were of interest, would support formal differential expression testing (e.g., with DESeq2 or edgeR).
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Gene annotation assigned function based on a fixed BLAST E-value cutoff (≤ 1e-5) against reference databases.↳ Could also: Reporting an FDR-adjusted significance threshold alongside E-value, or using bit-score/percent-identity cutoffs in combination — Because E-value already scales with database/search size, pairing it with an FDR-style summary or additional score metrics is a complementary way to communicate annotation confidence across many simultaneous comparisons.
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Summary results (e.g., percentages of SNP types, GO category proportions, primer validation success rate) are presented as point estimates without confidence intervals.↳ Could also: Bootstrap or binomial confidence intervals around proportions — Interval estimates would convey the precision of proportions such as the 31.4% primer validation success rate, which is based on a modest number of trials (51 primer pairs).
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Fifty-one SNP loci were selected for Tm-shift validation genotyping in 30 individuals, without a stated rationale for these specific sample and locus numbers.↳ Could also: A pre-specified sample-size/power calculation based on expected minor allele frequency and desired precision for heterozygosity estimates — This would document how the chosen numbers of loci and individuals relate to the precision needed to characterize biallelic polymorphism, complementing the empirical selection approach used.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
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.
Input fidelity is perfect — SRR1023631 under SRP032493 yields exactly the paper's 58,944,478 raw reads, and SeqPrep+Sickle retention (98.53% incl. singletons, 97.17% paired-only) sits right on the reported 98.0%. The deviation is downstream and structural: the raw Trinity 2.3.2 assembly gives 217,399 transcripts / 160,054 genes with N50=483 bp and mean 443.74 bp, against the paper's 69,751 contigs, N50=1,127 bp, mean 928.8 bp — a gap best explained by an unnamed post-assembly redundancy-removal/clustering step the paper never documents, plus an unstated Trinity version. Whose side: shared between our method (we chose not to cluster) and the authors (the method as written is not complete enough to reproduce those figures); there is no indication of fabrication. Severity is moderate: the qualitative resource claim survives — 4,222 SSRs (87% of 4,871) with trinucleotides dominant (76.4% vs 92.4%, tri >> di > tetra > penta/hexa) — but the headline assembly-quality numbers do not reproduce and downstream contig-based statements should not be assumed to carry over.
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