A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum.
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.
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
Reproduced the three named pipelines (RNA-seq TopHat+Cufflinks+HTSeq+DESeq2; WGBS BS-Seeker2 methylation calling; CRI/RIP repeat analysis via critool+RepeatMasker) from PMID 25392735 end-to-end on all 9 GSE49700 samples (5 WGBS, 4 RNA-seq). RNA-seq raw read counts match the paper exactly for all 4 samples; unique-mapping rate matches closely for FLM/FLM_aza/FLM_untreated but is notably lower than reported for FB. WGBS genome-wide %methylation reproduces well for CG context in most samples and very well for FLM_aza across all 3 contexts, but shows a systematic upward inflation in CHG/CHH for FB/FLM/FLM_untreated and a large, consistent mismatch for ECM across all 3 contexts. CRI/RIP results are qualitatively consistent with the paper's claim that T. melanosporum shows a weaker RIP-like signature than N. crassa, though the paper gives no numeric CRI value to match exactly. Cufflinks novel-gene counts are consistently ~2.3-3x higher than reported for all 4 samples (same direction, different magnitude). DESeq2 differential expression (5-aza treated vs untreated, via an approximate no-replicate workaround) found 139 significant genes vs the paper's reported 115 -- same order of magnitude. WG-seq (whole-genome resequencing) was correctly excluded as out of scope. All large reproducible intermediates (~208GB: raw/trimmed reads, BAM/CGmap/ATCGmap, conda pkg caches, RepeatMasker scratch output) were deleted after result extraction; curated small artifacts and logs are retained under scratch/pmid-25392735/curated/ and logs/.
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- Reproduced
- 2026-08-05
- Rubric version
- not recorded
- Assessed by
- —
- 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: opusThe paper asks if and how the black truffle Tuber melanosporum, which has one of the largest and most transposable-element-rich fungal genomes (125 Mb, >58% repetitive DNA, >45,000 repeated elements), exploits DNA methylation to silence transposable elements and maintain genome integrity, and whether this methylation is reversible and linked to transcription.
- ★ T. melanosporum shows a high rate of cytosine methylation (>44%) that selectively targets transposable elements rather than genes, with a strong preference for CpG sites. finding
- ★ Silenced TEs are highly methylated whereas expressed TEs show an anti-correlation between methylation and transcription, indicating TE expression is regulated by DNA methylation; gene-body methylation shows no relationship with gene expression level. mechanism
- ★ Whole-genome sequencing uncovered multiple TE-enriched copy number variant regions containing a significant fraction of hypomethylated and expressed TEs, almost exclusively in in vitro-propagated free-living mycelium. finding
- ★ Treatment of mycelia with the demethylating agent 5-azacytidine partially reduced DNA methylation and increased TE transcription, indicating a non-exhaustive and partly reversible methylation process. finding
- ★ A comprehensive BS-seq, WG-seq and RNA-seq dataset across fruitbody, free-living mycelium and ectomycorrhiza developmental stages is provided as a community resource for evolutionary (epi)genomics of Pezizomycotina. resource
- ★ Transcriptome assembly identified 614 novel genes not present in the original truffle v1.0 annotation, including 68 novel genes/transcripts present only in 5-aza treated mycelium. finding
- Genes differentially expressed between untreated and 5-aza treated mycelia are enriched for oxidation-reduction processes and transmembrane transport, supporting an epigenetic regulatory system responsive to environmental stress. finding
- An implementation of the Composite Repeat Induced Point mutation index (CRI), a dinucleotide frequency distribution tool for assessing RIP-induced methylation, is provided. method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Whole-genome bisulfite sequencing (BS-seq) | Tuber melanosporum Mel28 strain free-living mycelium (grown 5 weeks on 1% malt agar) and field-collected mature fruitbody | none | Per-cytosine methylation level #C/(#C+#T) in CG/CHG/CHH contexts; genome-wide methylation profiles and read-depth-based CNV | Illumina HiSeq 2000; EpiTect bisulfite kit (QIAGEN); DNeasy Plant Mini kit; BS Seeker 2 alignment to Tuber_melanosporum_v1.0 |
| Whole-genome bisulfite sequencing (BS-seq) | Ectomycorrhizal (ECM) root tips of common hazel (Corylus avellana L.) plantlets inoculated with T. melanosporum mycelium slurry | symbiotic association (inoculation) | Low-resolution global CG/CHG/CHH methylation profiles of the symbiotic stage (1.18X per-strand coverage) | Illumina HiSeq 2000; BS Seeker 2 |
| Whole-genome bisulfite sequencing (BS-seq) | T. melanosporum mycelia grown in the dark at 23 °C in synthetic liquid medium | 5-azacytidine (drug) added every 5 days at 10, 40 and 100 μM final concentration for 45 days, versus water-added control | Genome, gene and TE methylation levels in CG/CHG/CHH contexts in treated vs untreated mycelia | Illumina HiSeq 2000; EpiTect kit (QIAGEN) |
| Whole-genome (non-bisulfite) DNA sequencing (WG-seq) | T. melanosporum free-living mycelium and fruitbody genomic DNA | none | Read-depth coverage used to independently confirm copy number variant regions called from BS-seq | Standard Illumina sequencing (51-bp reads) |
| mRNA sequencing (RNA-seq) | T. melanosporum fruitbody and free-living mycelium | none | Read counts per gene and per TE; expression levels as RPKM; coverage depth per gene/TE | Illumina TruSeq RNA Sample Preparation kit, Illumina HiSeq 2000, 50-bp single-end reads; RNeasy Plant Mini kit; Bioanalyzer (Agilent); Qubit RNA BR Assay kit; TopHat/HTSeq v0.5.4p3/FastQC |
| mRNA sequencing (RNA-seq) | T. melanosporum free-living mycelium in synthetic liquid medium | 5-azacytidine treatment (10, 40, 100 μM over 45 days) vs untreated control | Differential gene expression (RPKM, fold change, adjusted p value), TE transcription, functional enrichment via Blast2GO, and treatment-specific novel transcripts | Illumina, 51-bp reads; Blast2GO |
| Guided transcriptome assembly / novel gene prediction (computational) | RNA-seq data from FB, FLM, 5-aza treated and untreated FLM mapped to truffle v1.0 reference assembly (7,496 genes) | none | Number of novel intergenic transcripts (Cuffcompare class code 'u', FPKM ≥4 in the 95% confidence interval) clustered into novel genes with BlastX protein homology | TopHat, Cufflinks, Cuffcompare, Blast2GO (BlastX) |
| Composite Repeat Induced Point mutation index (CRI) dinucleotide frequency analysis | T. melanosporum genome sequence | none | Likelihood that genome DNA methylation is induced by Repeat-Induced Point mutation (RIP) | — |
- ▲ Cytosine methylation rate across the T. melanosporum genome is high (>44%), selectively targeting TEs rather than genes with a strong preference for CpG sites. >44%
- – TEs are heavily CG-methylated (70.51% FLM, 69.71% FB, 59.16% ECM) while genes are almost devoid of methylation (CG 0.64% FLM, 0.87% FB, 0.84% ECM). TE CG 70.51% vs gene CG 0.64% (FLM)
- – Genome-wide CG methylation was 30.3% (FLM), 28.9% (FB) and 6.4% (ECM); CHG and CHH levels were much lower (8.1-10.3% in FLM/FB). CG 30.3%/28.9%/6.4%
- – Genes are methylation-depleted in all contexts with no difference between high, medium and low expression classes, whereas silenced TEs are 70-80% methylated and expressed TEs show clear methylation/transcription anti-correlation. silenced TEs 70-80% methylated
- – 107 genomic regions with significant CNV between FLM and FB (100 kb windows with |log coverage ratio FLM/FB| ≥ 0.3) were identified, covering 7.3% of the genome; 102 (95%) were independently confirmed by non-bisulfite Illumina WGS of FLM DNA. 107 regions, 7.3% of genome, 95% confirmed
- – A total of 614 novel genes were identified after merging assemblies (282 in FB, 328 in FLM, 466 in 5-aza treated, 440 in 5-aza untreated), following removal of 11 extremely long genes (≥36,573 bp). 614 novel genes
- ▲ 68 novel genes/transcripts were detected only in 5-aza treated mycelium and not in untreated mycelium. 68 novel genes/transcripts
- – Oxidation-reduction and transmembrane transport genes were strongly induced by 5-aza, e.g. flavin-binding monooxygenase-like protein (RPKM 8 → 258) and extracellular dioxygenase (RPKM 18 → 196), while others such as MFS drug efflux were repressed (RPKM 663 → 84). 33.80-fold up; 10.71-fold up; 0.13-fold (down)
- fold_change 33.80 (GSTUMT00004482001 flavin-binding monooxygenase-like protein, 5-aza treated vs untreated mycelium (RPKM 8 → 258), adj. p = 3.51E-09)
- pvalue 4.71E-13 (Lowest reported adjusted p value; GSTUMT00001850001 endo-beta-glucanase eng1, 16.02-fold up in 5-aza treated mycelium)
- fold_change 0.10 (GSTUMT00007117001 D-isomer specific 2-hydroxyacid dehydrogenase, most strongly down-regulated by 5-aza (RPKM 4 → 0.4), adj. p = 4.66E-02)
- count 107 CNV regions corresponding to 7.3% of the genome; 102 (95%) confirmed by WGS (Copy number variant regions between FLM and FB called from BS-seq read depth (100 kb windows, |log ratio| ≥ 0.3))
- count 614 (Novel genes identified from the merged RNA-seq transcript assembly against truffle v1.0 (7,496 annotated genes))
- other 31.61X (FLM), 34.73X (FB), 1.18X (ECM), 8.44X (5-aza treated), 7.21X (5-aza untreated) coverage per strand (BS-seq library coverage; ~90% of cytosines retained for FB/FLM analysis and ~75% for 5-aza samples (coverage ≥4 reads))
- count 63,742,213 (FB) and 74,109,065 (FLM) uniquely mapped RNA-seq reads, 93.34% and 84.88% overall mapping (RNA-seq alignment statistics for fruitbody and free-living mycelium)
- other RIN 7.0 (FB) and 6.5 (FLM); ~75% of genes covered at ≥10 reads in FB/FLM, ~90% in 5-aza samples; 55-60% of TEs covered in both samples per comparison (RNA quality and RNA-seq coverage completeness metrics)
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 data-note resource paper describing whole-genome bisulfite sequencing (BS-seq), whole-genome sequencing (WGS) and RNA-seq of Tuber melanosporum across developmental stages (FLM, FB, ECM) and 5-azacytidine treated/untreated mycelium, apparently one sample per condition. Differential gene expression between untreated and 5-aza treated mycelia is reported as RPKM fold-change with an adjusted p-value per gene (Table 5), and novel transcripts were called from Cufflinks/Cuffcompare assemblies using an FPKM threshold with a 95% confidence interval. Copy-number variant regions were defined using a fixed log-coverage-ratio cutoff, and results are otherwise reported as summary percentages/coverage statistics rather than through classical group-comparison tests.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| not stated (differential expression reported as fold change with an adjusted p-value) | Table 5, genes differentially expressed between untreated and 5-aza treated mycelia | — | not stated |
| FPKM 95% confidence interval threshold (Cufflinks/Cuffcompare) used to call novel transcripts | novel gene/transcript prediction from RNA-seq assemblies | — | not stated |
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Differential expression between untreated and 5-aza treated mycelia was summarized as fold change with an adjusted p-value, without the underlying statistical/count model being named in the text.↳ Could also: A count-based RNA-seq differential expression tool such as DESeq2 or edgeR could also be used — These explicitly model read-count overdispersion and provide a documented statistical framework (e.g., negative-binomial Wald or likelihood-ratio tests) alongside the fold-change and adjusted p-value already reported.
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Comparisons across developmental stages and treatment appear to be based on one sample per condition rather than multiple biological replicates.↳ Could also: Designs with multiple biological replicates per condition, analyzed with replicate-aware methods (e.g., DESeq2/edgeR or ANOVA), could also be used — Replicates allow estimation of biological variability and would let dispersion (e.g., SD, CI) be reported alongside the point estimates already given.
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Multiple testing correction was applied to the differential expression results (an 'adjusted p value' is reported) without naming the specific method.↳ Could also: Explicitly stating and applying a named method such as Benjamini-Hochberg FDR or Bonferroni could also be used — Naming the method makes the stringency of the correction and the assumptions behind it transparent and reproducible.
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Novel transcripts were defined using a fixed FPKM threshold (>=4) within a 95% FPKM confidence interval from Cufflinks/Cuffcompare.↳ Could also: A permutation-based or model-based transcript-confidence approach (e.g., as implemented in Cuffdiff or a Bayesian expression caller) could also be used — Such approaches can provide an alternative, model-based estimate of calling confidence that complements a fixed FPKM cutoff.
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Copy-number variant regions were defined using a fixed threshold (|log2 coverage ratio FLM/FB| >= 0.3 over 100 kb windows) rather than a formal statistical test.↳ Could also: A dedicated CNV-calling method with statistical significance testing (e.g., circular binary segmentation, as in DNAcopy) could also be used — Such methods provide segment-level significance or confidence estimates in addition to a coverage-ratio threshold, which can complement the concordance check already performed against standard Illumina sequencing.
What was reproduced
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