GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring.
The main results reproduced, with only marginal, non-material deviations.
- Nothing in this column.
- 🔴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 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
PROVISIONAL/IN-PROGRESS. Cited code = pyani (ANI of 23 GAL08 SAGs+MAGs); headline result (3 species; within >99.2% / between <88.1% ANI; 95-96% delineation; genome stats 3.17 Mb / 62.8% GC) depends on the 23 assembled genomes which are JGI IMG registered-access only (HTTP 403 anonymous; not mirrored at NCBI; no JGI account available) -> not runnable on the authors' exact data (data_restricted for C1-C3,C7-C8). The cited OPEN accession PRJNA779083 is 32 16S amplicon runs supporting a different pipeline (QIIME) for the abundance claims C4-C6; these are attemptable. Only 7/23 SAGs have open raw reads, allowing at best a partial deviating self-assembly+pyani corroboration. Heavy compute deferred: «our HPC»/VPN down at start (port 22 timeout) - waiting per protocol. This file will be refined once compute runs.
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Assessment versions
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v1 current initial assessment Score 50assessed: 2026-06-19 ⛓ a08ac43942a3
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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
- 2026-06-19
- 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
- 2026-07-31
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: opusWhat is the abundance, distribution, and genomic/metabolic potential of the uncultivated GAL08 clade of Acidobacteria in a pH-neutral hot spring (Dewar Creek, British Columbia), and what is its phylogenetic placement and lifestyle?
- ★ GAL08 is a dominant bacterial clade in a neutral hot spring, comprising up to 29.2% of the community by relative read abundance and up to 4.7 × 10^5 16S rRNA gene copies per gram sediment. finding
- ★ Absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. finding
- ★ The three GAL08 bacteria represent a distinct subgroup of Acidobacteria, a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. finding
- ★ GAL08 genomes predict a heterotrophic metabolism with potential for aerobic respiration, incomplete denitrification, and fermentation. mechanism
- ★ GAL08 appears to have a microaerophilic lifestyle: qPCR counts declined rapidly under atmospheric oxygen but increased slightly at 1% (v/v) O2. finding
- ★ 25 SAGs and 7 MAGs were generated representing three separate species based on ANI, with average genome size 3.17 Mb and GC content 62.8%. resource
- A GAL08-specific qPCR primer set was developed to detect the GAL08 16S rRNA gene. method
- GAL08 was a consistently dominant clade across multiple sampling years and locations. finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| 16S rRNA gene amplicon sequencing (Illumina MiSeq) | Dewar Creek hot spring sediment, 22.5–79.8°C | none | relative abundance of GAL08 (OTUs/ASVs) in community | Illumina MiSeq, MiSeq Reagent Kit v3 600 cycles; primers 341fw/785rv |
| qPCR of 16S rRNA genes (GAL08-specific) | Dewar Creek and Hoodoo Creek hot spring sediment | none | absolute 16S rRNA gene copy number per gram sediment | Rotor-Gene 6000, SYBR Green qPCR master mix; primers GAL08_842/GAL08_992 |
| Single amplified genome (SAG) generation via single-cell sorting/WGA/sequencing | Dewar Creek sediment supernatant, samples at 65 and 77°C | none | genome assembly, completeness, ANI, metabolic gene content | FACS, SPAdes assembly, ProDeGe QC |
| Metagenome sequencing and MAG reconstruction | Dewar Creek sediment at 45, 65, 66, 77°C | none | binned genomes (MAGs), completeness, contamination | IMG/JGI (CheckM); IMG IDs 3300025105, 3300025094, 3300025775, 3300006767 |
| Phylogenetic reconstruction (16S rRNA Bayesian tree; 56-marker concatenated ML tree) | DChs_GAL08 sequences vs Acidobacteria/reference genomes | none | phylogenetic placement of GAL08 | MrBayes v3.2.6; IQ-TREE; MAFFT; HMMER; Prodigal |
| Fluorescence in situ hybridization (FISH) | Hoodoo Creek hot spring sediment, 61°C | none | in situ detection/visualization of GAL08 cells | probes GAL08_185_cy3 and EUB338_Alexa488 |
| Laboratory cultivation/enrichment with varied O2 | Dewar Creek sediment (2017 samples) | oxygen level (atmospheric vs 1% v/v O2) | GAL08 abundance change by qPCR | — |
| Genome annotation and comparative analysis (ANI, COG, iron genes) | GAL08 SAGs and MAGs | none | metabolic pathway prediction, core/unique COGs, species delineation | IMG/JGI; pyani; FeGenie; CheckM |
- – GAL08 relative and absolute abundance peaked at 65°C across three temperature transects
- ▲ GAL08 comprised up to 29.2% of the microbial community by relative read abundance 29.2%
- ▲ GAL08 reached up to 4.7 × 10^5 16S rRNA gene copies per gram sediment by qPCR 4.7 × 10^5 copies/g
- – SAGs and MAGs represented three separate species with average genome size 3.17 Mb and GC content 62.8% 3.17 Mb; 62.8% GC
- – GAL08 qPCR counts declined rapidly under atmospheric O2 but increased slightly at 1% O2
- – GAL08 placed as a distinct subgroup within class Blastocatellia (candidate order Ca. Frugalibacteriales)
- – GAL08 detected at Hoodoo Creek at 3.8 × 10^4 gene copies per gram sediment by qPCR 3.8 × 10^4 copies/g
- – DChs_GAL08 shows closest 16S rRNA gene identity (82–84%) to other phyla (Thermotogae, Aquificae, etc.) 82–84% identity
- count 29.2% (maximum GAL08 relative read abundance in community)
- count 4.7 × 10^5 16S rRNA gene copies per gram sediment (maximum GAL08 abundance by qPCR at Dewar Creek)
- count 3.8 × 10^4 gene copies per gram sediment (GAL08 abundance at Hoodoo Creek (61°C))
- mean 3.17 Mb (estimated average genome size of three GAL08 species)
- mean 62.8% (average GC content of GAL08 genomes)
- count 25 SAGs and 7 MAGs (genomes recovered identified as GAL08)
- other completeness 97.2% to 14.2% (estimated completeness range of SAGs (CheckM))
- other 82–84% (16S rRNA gene identity of GAL08 to closest reference phyla)
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 a descriptive environmental microbiology and comparative genomics study characterizing the uncultivated GAL08 clade in a neutral hot spring. Abundance was quantified using 16S rRNA amplicon sequencing (relative abundance) and qPCR (absolute gene copy numbers per gram sediment) across temperature gradients and multiple sampling years (2010–2017). Phylogenetic placement was established using Bayesian inference (MrBayes) and maximum-likelihood (IQ-TREE) methods on 16S rRNA and concatenated marker gene datasets. No formal inferential hypothesis tests (e.g., ANOVA, t-tests) were performed; all quantitative results are reported descriptively.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Bayesian MCMC phylogenetic inference (MrBayes, GTR+Gamma+I substitution model) | 16S rRNA gene-based phylogenetic tree for Acidobacteria placement | 153 sequences of ≥1,395 bp; 2×10^6 MCMC iterations, burn-in 1×10^5 | stated |
| Maximum-likelihood phylogenetic inference with bootstrap support (IQ-TREE, 1,000 bootstraps) | Concatenated 56-marker gene tree for genomic placement of GAL08 within Acidobacteria and broader bacterial phyla | 25 SAGs/7 MAGs + 68 Acidobacteria genomes + 214 reference genomes; 6 Parcubacteria as outgroup | not stated |
| OTU clustering at 97% similarity with BLAST taxonomic classification against Silva 119 (QIIME 1.9.1) | Relative abundance of GAL08 in the broader microbial community from 16S rRNA amplicon libraries | 32 samples collected 2012–2015 | not stated |
| ASV denoising via DADA2 with Silva 138 classification (QIIME2 2021.4) | Identification of all DChs_GAL08 amplicon sequence variants | 32 samples collected 2012–2015 | not stated |
| Average nucleotide identity (ANI) pairwise comparison (pyani) | Species-level grouping of SAGs and MAGs using a standard 95% ANI threshold | 25 SAGs and 7 MAGs | not stated |
| SYBR Green qPCR quantification against a dilution series standard curve (Rotor-Gene 6000) | Absolute abundance of DChs_GAL08 16S rRNA gene copies per gram sediment across temperature gradients and years | Samples from field trips in 2011, 2015, and 2017; number of technical replicates not stated | not stated |
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qPCR absolute abundance was reported as single point values (e.g., up to 4.7×10^5 gene copies per gram sediment) without any dispersion measure↳ Could also: Report mean ± SD or mean ± SEM across technical replicates, or provide 95% confidence intervals derived from the standard curve regression — Dispersion measures communicate measurement variability and allow readers to judge reproducibility; for qPCR data, reporting variability across technical or biological replicates is standard practice and aids interpretation of abundance differences across temperatures or years
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The relationship between temperature and GAL08 abundance was described qualitatively (peaked at ~65°C along three transects)↳ Could also: Fit a unimodal response model (e.g., Gaussian curve, second-degree polynomial regression, or a GAM) to the temperature–abundance data — A formal model would estimate the thermal optimum and niche width with confidence intervals, enabling quantitative comparison across sampling years and stream transects and placing the result in the context of other thermophile ecology studies
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Two parallel amplicon analyses were run (OTU-based in QIIME 1.9.1; ASV-based in QIIME2/DADA2), apparently for different purposes↳ Could also: Use ASVs (DADA2 or Deblur) as the single primary abundance unit throughout, replacing the 97% OTU clustering step — ASVs provide single-nucleotide resolution, are fully reproducible across studies, and avoid the arbitrary 97% threshold that can merge ecologically distinct populations; the paper already uses DADA2, so unifying on ASVs would harmonize both analyses
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Relative community composition from amplicon data was reported descriptively without diversity metrics or statistical comparison across temperature bins or sampling years↳ Could also: Calculate alpha-diversity indices (e.g., Shannon entropy, observed ASVs after rarefaction) and perform beta-diversity ordination (Bray-Curtis PCoA) with a permutation test (PERMANOVA/adonis) to test temperature or year as predictors — Ordination and PERMANOVA would quantify and statistically support the observed temperature-driven community structuring, making the pattern more directly comparable with other environmental gradient studies
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Genome completeness and contamination were estimated using CheckM with a universal single-copy marker gene set↳ Could also: Cross-validate with a taxon-specific CheckM lineage workflow or with BUSCO using a relevant lineage database — Universal marker sets may be under-represented or biased for deeply divergent, uncultivated lineages; a taxon-specific or complementary completeness tool would provide an independent quality estimate tailored to this novel Acidobacteria clade
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Species-level delineation of SAGs and MAGs relied solely on pairwise ANI with a 95% threshold↳ Could also: Supplement ANI with digital DNA–DNA hybridization (dDDH via the GGDC server) or tetranucleotide frequency-based clustering — Using multiple converging species delineation methods strengthens candidate species proposals; dDDH in particular is a widely accepted complement to ANI in formal prokaryotic taxonomic descriptions and provides an independent probability-based similarity estimate
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-35087491
Paper: Ruhl et al. 2022, GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring. Front Microbiol 12:787651. PMID 35087491 · PMCID PMC8787282 · DOI 10.3389/fmicb.2021.787651
Cited code: https://github.com/widdowquinn/pyani (third-party ANI tool — valid per P16)
Cited data: SRA PRJNA779083 (amplicon 16S rRNA sequencing)
Datasets the paper relies on
| dataset | what | access | used for |
|---|---|---|---|
PRJNA779083 (SRA) |
32 Illumina 16S amplicon runs (341f/785r), 2012–2015 temperature transects | OPEN | abundance / QIIME pipeline (C4–C6) |
| IMG SAG/MAG genomes (Table 1 taxon IDs) | 16 SAGs + 7 MAGs assembled genomes of DChs_GAL08 | JGI IMG only — registered access (no anonymous download; taxon-detail pages HTTP 403; no JGI account in secret store) | pyani ANI, genome stats, marker-gene tree (C1–C3, C7–C8) |
| GAL08 SAG raw reads (PRJNA469195/469184/469179, PRJNA364518/364596/364633/364655) | 7 single-cell raw read sets ("hot springs metagenome", library OTHER) | OPEN (SRA/ENA) | only the raw reads for 7 of the 23 SAGs; the assembled genomes used by the paper are IMG-only; all 7 MAGs have no open product |
Key mismatch: the cited code (pyani) operates on the assembled genomes, which are deposited in JGI IMG (registered access), while the cited open accession (PRJNA779083) is amplicon data for a different pipeline (QIIME). The assembled genomes are not in NCBI GenBank/WGS (assembly search for "GAL08" = 0 hits; data availability statement: "publicly available in the IMG database").
Reproducible results
In scope — OPEN data (attemptable)
- C4 GAL08 relative abundance up to 29.2 %; 7.0–29.2 % across the 15 samples at 64.3–67.4 °C; community peak ~65 °C. (Results; Fig 1/2) — pipeline: QIIME2 on PRJNA779083.
- C5 ASV1 = on average 95.9 % of reads identified as DChs_GAL08. (Results; Supp Fig 6) — QIIME2.
- C6 At 60–85 °C GAL08 = on average 89.0 % of acidobacterial reads. (Results) — QIIME2.
In scope — but data RESTRICTED (cannot run on authors' genomes)
- C1 (headline) pyani ANI of all SAGs+MAGs → 3 species-level clusters; species delineation 95–96 % ANI; within-cluster >99.2 %, between-cluster <88.1 %. (Fig 3A) — needs the 23 IMG genomes.
- C2 estimated average genome size 3.17 Mb, GC 62.8 %. (Abstract/Results) — IMG genomes.
- C3 16S of Species 1 vs 3 differ by 0.19 % (full-length). (Results) — IMG genomes.
- C7 80.2 % average 16S divergence to cultivated Blastocatellia. (Results; Fig 5) — IMG genomes + refs.
- C8 56-concatenated-marker-gene tree, 1000 bootstraps; 153-seq 16S tree. (Fig 4/5) — IMG genomes + refs (heavy).
Partial illustrative path (OPEN raw reads, deviation)
- 7 GAL08 SAG raw-read sets are open → self-assemble (SPAdes) + run pyani to test whether the open subset reproduces the within/between-species ANI structure of C1. Clearly a deviation (self-assembly vs JGI assembly) and incomplete (7/23, no MAGs), so at best a partial corroboration of the headline.
Out of scope (wet-lab / manual / interpretive)
qPCR 16S copy numbers (4.7×10⁵/g), temperature measurements, FISH, metabolic reconstruction interpretation, candidate taxonomy naming.
Blockers
- «our HPC»/VPN down at start (
«host».«infra».uni-hamburg.de:22connection timed out). Waiting per protocol; not touching the VPN. All heavy compute deferred until tunnel returns. - IMG registered access for the 23 assembled genomes → headline ANI not runnable on the authors' exact data.
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Reproduction footprint
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