Metatranscriptomics From a Small Aquatic System: Microeukaryotic Community Functions Through the Diurnal Cycle.
Part of the results reproduced; minor but material deviations remained.
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- ✓Same input data as the authors
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
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- 🟡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
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- Reproduced
- 2026-06-19
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-18no human curator yet
- Last updated
- 2026-07-29
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: opusDoes the microeukaryotic community of a small temperate freshwater pond regulate its metabolic processes in response to the diurnal light/dark cycle in the same way that has been reported for larger aquatic systems (open oceans and large lakes)?
- ★ Photosynthesis-related and translational transcripts are upregulated at midday (high light) compared to night/darkness in the pond microeukaryotic community finding
- ★ Unique GO classes characterize each condition: day favors photoreception/photosynthesis, defense and stress mechanisms, while night favors motility, ribosomal assembly and other energy-consuming processes finding
- ★ The small pond community follows diurnal expression dynamics similar to those described for larger aquatic ecosystems finding
- ★ Euglenophyta and Chlorophyta dominate the active phototrophic community of the pond finding
- ★ Combining pigment analyses (HPLC/CHEMTAX), metatranscriptomics, and physicochemical data provides greater insight into microeukaryotic metabolic processes than any single method method
- Metatranscriptomics alone cannot determine taxonomic composition of an active community and requires complementary pigment-based approaches method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Metatranscriptomics / RNA-Seq (poly-A selected mRNA, de novo transcriptome assembly, differential expression) | Microeukaryotic community from a small artificial freshwater pond, Botanical Gardens, University of Cologne, Germany | Diurnal cycle (day/midday vs night sampling) | Transcript abundance, differentially expressed transcripts, GO term enrichment | Illumina HiSeq 4000, 75 bp paired-end; TruSeq stranded library kit |
| Accessory photopigment analysis by HPLC + CHEMTAX | Phytoplankton/phototrophic community of the pond seston | Diurnal cycle (day vs night) | Pigment composition and relative contribution of phytoplankton groups to total chlorophyll a | Shimadzu Prominence HPLC system, Spherisorb ODS2 C18 column |
| In situ physicochemical measurement | Pond water | Diurnal cycle (day vs night) | Temperature, pH, PAR (air and water), dissolved oxygen, conductivity | WTW pH meter Vario; LI-COR LI-193 quantum sensor; Hach HQd optical DO sensor; WTW LF330 conductivity meter |
| Particulate carbon/nitrogen elemental analysis | Pond seston (GF/F filtered) | Diurnal cycle (day vs night) | POC, PON, C:N ratios | Thermo Flash EA2000 Analyzer |
| Soluble reactive phosphorus spectrophotometry (ascorbic acid molybdenum-blue) | Filtered pond water | Diurnal cycle (day vs night) | SRP concentration | Hach DR5000 UV-Vis spectrophotometer |
| Dissolved organic carbon / fluorescence analysis | 0.2 µm filtered pond water | none | DOC concentration and humification/biological indices (bix, fI, hix) | HORIBA Aqualog fluorometer; staRdom R package |
- ▼ Chlorophyll a content significantly higher during day than night 3.63 vs 1.46 µg/L
- ▲ Differentially expressed transcripts (photosynthesis and translation related) upregulated at midday vs night
- ▲ Day-unique GO terms dominated by photosynthesis, protein-chromophore linkage, defense response to bacteria, and stress/cold response
- ▲ Night-unique GO terms dominated by ribosome biogenesis, ribosomal small subunit assembly, cilium/dynein motility and meiotic cell cycle
- – Euglenophytes and Chlorophytes were the most abundant phototrophic groups in almost all samples; relative abundance of most groups (esp. Chrysophytes, Cryptophytes, Cyanobacteria) decreased at night
- ▲ More transcripts identified in day metatranscriptome than night 763,363 (day) vs 509,532 (night) transcripts
- – POC and dissolved oxygen significantly higher during day; PON, C:N, pH, conductivity, SRP not significantly different POC 1.79 vs 1.25 mg/L
- – GO terms: 4693 shared between day and night, 5101 unique to day, 4388 unique to night
- count 763,363 day transcripts; 509,532 night transcripts (Total transcripts identified after annotation in day vs night metatranscriptomes)
- count 14,792,542 to 16,756,988 reads per sample (Sequencing yield per sample)
- pvalue P < 0.001 (Chlorophyll a difference between day (3.63±1.65) and night (1.46±0.99) µg/L)
- mean PAR air day 1999.38±73.66 vs night 0.12±0.08 µmol s-1 m-2 (PAR in air, day vs night, P<0.001)
- pvalue P < 0.05 (POC day 1.79±0.52 vs night 1.25±0.56 mg/L significant difference)
- count 4693 shared, 5101 day-unique, 4388 night-unique GO terms (Gene ontology terms by condition)
- mean DOC 11.3 g/L (bix=0.681; fI=1.38; hix=0.887) (Dissolved organic carbon concentration and fluorescence indices)
- other ~15% of transcripts annotated as proteins; ~40% identified as potential proteins (Annotation success against Swissprot database)
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 compared microeukaryotic gene expression and physicochemical parameters between midday and nighttime samples collected on 4 separate days from a single small freshwater pond, treating each day as a biological replicate per condition. Physicochemical and pigment data were analyzed with classical frequentist tests (Shapiro-Wilk, t-tests or Mann-Whitney, two-way ANOVA with Tukey post-hoc), while RNA-seq differential expression was analyzed with edgeR (negative binomial, TMM normalization, Benjamini-Hochberg FDR) and GO enrichment with GOseq (random-resampling null distribution, FDR 0.05). Results for physicochemical parameters were summarized as mean ± SD and significance was reported as categorical thresholds rather than exact p-values.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Shapiro-Wilk normality test | All physicochemical parameters, used to select between t-test and Mann-Whitney | n=4 per condition for in situ parameters; n=12 per condition for triplicate nutrient analyses | na |
| Student's t-test (two-sample) | Day vs. night comparison of physicochemical parameters (for normally distributed data) | n=4 per condition for in situ parameters; n=12 per condition for triplicate nutrient analyses | not stated |
| Mann-Whitney rank-sum test | Day vs. night comparison of physicochemical parameters (for non-normally distributed data) | n=4 per condition for in situ parameters; n=12 per condition for triplicate nutrient analyses | not stated |
| Two-way ANOVA (factors: day/night and phyla) | Relative and absolute pigment composition across sampling dates and phyla | n=4 sampling days per condition | not stated |
| Tukey HSD post-hoc pairwise comparisons | Following two-way ANOVA on pigment composition, comparing different sampling dates and phyla | n=4 sampling days per condition | not stated |
| edgeR negative binomial model with TMM normalization and Benjamini-Hochberg FDR (adjusted p < 0.001) | Transcript-level differential expression between day and night metatranscriptomes | n=4 biological replicates per condition | not stated |
| GOseq enrichment test with random resampling null distribution and FDR 0.05 | Gene Ontology enrichment of over-represented differentially expressed transcripts | count of DE transcripts not stated | not stated |
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Multiple physicochemical and nutrient parameters were each tested individually (t-test or Mann-Whitney) without a stated correction across that family of simultaneous tests↳ Could also: A family-wise correction (e.g., Holm-Bonferroni) across the set of univariate tests, or a permutation-based multivariate test such as PERMANOVA on the full parameter matrix — Applying a correction across the family of simultaneous tests controls the probability of at least one false positive; a multivariate test additionally accounts for correlations among environmental parameters and is commonly used in community ecology studies
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Each of the 4 sampling days contributed both a midday and a nighttime sample, and these were analyzed as independent replicates↳ Could also: Paired t-tests or Wilcoxon signed-rank tests, treating each calendar day as a matched pair (midday vs. night from the same day) — When the same sampling occasion contributes both levels of a factor, a paired design can account for within-day temporal correlation in environmental conditions and can increase statistical power relative to an unpaired approach
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Transcript-level differential expression was analyzed with edgeR (negative binomial, TMM normalization)↳ Could also: DESeq2 (negative binomial with median-of-ratios size-factor estimation) or limma-voom (log-CPM with observation-level weights) — DESeq2 and limma-voom are widely used alternatives that differ in their dispersion-estimation strategies and normalization approaches; applying multiple methods and comparing overlapping results is a common sensitivity check in low-replicate RNA-seq studies
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GO enrichment was assessed with GOseq using a random-resampling null distribution↳ Could also: topGO (which exploits the hierarchical GO graph to reduce redundancy among related terms) or clusterProfiler (which offers both over-representation and gene-set enrichment algorithms) — Different enrichment tools handle gene-length bias, GO-graph topology, and background-set definition differently; comparing results across tools can reveal which findings are robust across methodological choices
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Physicochemical results were summarized as mean ± SD with n=4↳ Could also: 95% confidence intervals, or display of individual data points alongside the mean — With four replicates, 95% CIs communicate both the spread and the estimation uncertainty of the mean; showing individual data points is increasingly recommended for small-n studies to improve transparency about the underlying data distribution
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Significance for physicochemical parameters was reported as categorical thresholds (P < 0.05, P < 0.001, N.S.) rather than exact p-values↳ Could also: Reporting exact p-values together with a standardized effect size measure (e.g., Cohen's d for t-tests, rank-biserial correlation for Mann-Whitney) — Exact p-values allow readers to apply their own thresholds and facilitate future meta-analyses; effect sizes convey the practical magnitude of differences independently of sample size, which is especially informative when n is small
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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Chlorophyll a concentration is significantly higher during the day (3.63 µg/L) than at night (1.46 µg/L) in a freshwater pond phytoplankton community.other freshwater-pond phytoplankton up 2020×1papers★ This paper is the founder (earliest)
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Relative contribution of most phytoplankton groups (especially Chrysophytes, Cryptophytes, Cyanobacteria) to total chlorophyll a decreases at night; Euglenophytes and Chlorophytes dominate across most samples.other freshwater-pond phytoplankton mixed 2020×1papers★ This paper is the founder (earliest)
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Dissolved oxygen and particulate organic carbon are significantly higher during the day than at night in a freshwater pond, consistent with daytime photosynthetic activity.other freshwater-pond water up 2020×1papers★ This paper is the founder (earliest)
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Defense response to bacteria, stress/cold response, and protein-chromophore linkage GO terms are uniquely enriched during the day in the freshwater pond microeukaryote metatranscriptome.RNA-seq freshwater-pond microeukaryote up 2020×1papers★ This paper is the founder (earliest)
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Photosynthesis and translation-related transcripts are upregulated at midday versus night in the freshwater pond microeukaryote metatranscriptome.RNA-seq freshwater-pond microeukaryote up 2020×1papers★ This paper is the founder (earliest)
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Ribosome biogenesis, ribosomal small subunit assembly, cilium/dynein motility, and meiotic cell cycle GO terms are uniquely enriched at night in the freshwater pond microeukaryote metatranscriptome.RNA-seq freshwater-pond microeukaryote up 2020×1papers★ 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.
Scope — pmid-32523568
Paper: Trench-Fiol & Fink (2020) Metatranscriptomics From a Small Aquatic System: Microeukaryotic Community Functions Through the Diurnal Cycle. Front Microbiol 11:1006. DOI 10.3389/fmicb.2020.01006.
Code: https://github.com/sjtf89/Cologne_pond (commit
c5d5070f65128fe58811362d7349d3eaa3101d7a, last push 2020-01-10).
The repo is data-only — it ships 7 derived-result files (no analysis
scripts). This is the P16 case: reproduction = (a) check the deposited derived
outputs against the paper's reported numbers, and (b) re-run the described
third-party pipeline on the paper's own raw data (SRA PRJNA596111).
Raw data: SRA BioProject PRJNA596111 = 8 runs SRR10716254–61 (4 day, 4 night biological replicates), 75 bp PE Illumina HiSeq 4000.
Pipeline described in Methods (the in-scope pipeline)
Trimmomatic 0.36 (LEADING:5 TRAILING:5 MINLEN:70, phred33) → SortMeRNA 2.1 (rRNA removal) → FLASH (read merging, ~30% merged) → Trinity 2.5.1 (de novo assembly, per condition) → Bowtie2 + RSEM (abundance, count matrices) → edgeR (TMM normalization, FDR cutoff) → Trinotate (TransDecoder ORFs, BlastX/BlastP vs SwissProt, HMMER vs PFAM) → GOseq (GO enrichment, 0.05 FDR).
In scope vs out of scope
| Result | Pipeline stage | In scope? | Approach |
|---|---|---|---|
| Physicochemical means±SD (Chl-a, POC, DO, water temp, PAR) | descriptive stats on deposited env CSV | YES (done) | recompute from Cologne_pond_physicochemical_parameters.csv |
| # DE (upregulated) transcripts: 45 total / 27 annotated | edgeR DE output (deposited) | YES (done) | count rows in deposited DEsubset files |
| Shared GO terms = 4,693 | GO comparison (deposited) | YES (done) | count GO IDs in GO_terms_shared_day_night.txt |
| Unique-day / unique-night GO = 5,101 / 4,388 | GO comparison (deposited) | YES (done — discrepant) | count GO IDs in deposited GO_terms_unique_*.txt |
| Raw reads/sample 14.79–16.76 M | SRA spot counts | YES (done — approx) | ENA read_run spot counts |
| rRNA contamination 5–10 % | Trimmomatic→SortMeRNA on raw reads | YES (running, «our HPC») | run described tools on SRR10716261 (day1) + SRR10716257 (night1) |
| Assembled transcripts: day 763,363 / night 509,532; ~223–331 Mb | Trinity de novo assembly | partially / not exact | de novo assembly is non-deterministic & version-sensitive; headline counts are not byte-reproducible by construction (documented, not forced) |
| 3 enriched (thylakoid) GO terms; 159 overrepresented (p<0.05) | GOseq enrichment | needs full assembly+annotation | depends on full count matrix + Trinotate background not shipped; deposited GO_terms_from_DE_transcripts.tsv lists 109 GO terms annotated to DE set |
Out of scope (not pipeline / wet-lab)
- Sampling, filtration, RNA extraction, environmental sensor measurement (wet-lab / field). Their summary statistics ARE reproduced (above).
- Ecological interpretation / community-function narrative (manual).
Assessments & scoring basis
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Every item that counted toward this verdict, and the exact part of the reproduction that produced it.
This is a P16 data-only consistency audit, and most of the paper's reported numbers are genuinely backed by the authors' deposit: all environmental statistics (C1–C9) and the DE/shared-GO counts (45 day-up, 0 night-up, 4,693 shared GO; C10–C13) recompute exactly to rounding. The one substantive defect is on the authors' side: the reported GO-unique counts (day 5,101 / night 4,388) are not derivable from the deposited unique_day/unique_night files (9,776 / 8,569 distinct GO IDs), which do not form a clean day/night/shared partition — a deposit/bookkeeping mismatch, not fabrication. A ~2% read-count discrepancy is a benign pairs-vs-mates convention issue. The central diurnal-differentiation conclusion stands (direction and DE backbone hold), so overall this is a solid, mostly 1:1 reproduction with two explainable, unverifiable GO counts.
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