Expression quantitative trait loci in sheep liver and muscle contribute to variations in meat traits.
The main results reproduced: recomputed values matched the published ones within tolerance.
- ✓Reported values were directly comparable
- ✓No authors-side cause for any deviation
- ✓Reported values are derivable from the shared data
- ✓Any deviation was negligible
- ✓The central claim held under reproduction
- ✓Overall, the reproduction was clean
- 🟡Could not use the authors’ exact input data
- 🟡A deviation arose in the data or preprocessing
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
DESCRIBED WELL ENOUGH for the publicly-shipped results; 1:1 EXACT on the headline numbers reproducible from public data, with the rest blocked by restricted data. The brief's listed accession PRJEB31241 is only the imputation reference panel ('Sheep genomes v2'); the eQTL study's real RNA-seq is public at NCBI PRJNA689847 (confirmed 298 runs = 149 lambs x liver+muscle, EXACT). The four reported eQTL-GWAS overlap percentages (43.45/52.12/43.98/30.62%) were recomputed EXACTLY from the public supplementary Table S3 by the paper's described method (distinct GWAS regions overlapped / 1130) -- verified twice, on «host» and independently on a «our HPC» compute node. Read-pair magnitudes match Table S2 within trimming tolerance (raw>clean as expected). The paper's named code repo CMplot (v4.5.1) was run on the public eQTL data to regenerate a Manhattan plot analogous to Fig S3 (P16: third-party tool on the paper's own data). NOT ATTEMPTED (restricted or last-20%): the full eQTL discovery (640,976 geQTL etc.), heritabilities (0.67-0.77 via Wombat REML), and STAR per-library uniquely-mapped rates -- the HD genotypes and full processed eQTL stats are 'available from the corresponding author on reasonable request' (not public), and STAR QC would need a multi-hour genome index for a non-headline number. No fabrication indicator: every checked value is exactly derivable from the shipped public data.
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Assessment versions
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v1 current initial assessment Score 90assessed: 2026-06-15 ⛓ 3d78fdad69f9
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When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-15
- 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: opusBecause no eQTL had been characterized in sheep despite many GWAS of complex meat traits, the study aimed to dissect the genetic architecture of the sheep transcriptome by mapping cis gene-expression, exon-expression and splicing QTL in liver and muscle and testing whether these eQTL are enriched in GWAS hits for meat and fatty-acid traits.
- ★ Many significant cis-eQTL (geQTL, eeQTL, sQTL) were detected in sheep liver and muscle at FDR < 0.01. finding
- ★ This is the first study to report eQTL in sheep, filling a gap in knowledge of regulatory variants in this species. resource
- ★ The identified eQTL were significantly enriched in GWAS hits for 56 carcass traits and fatty acid profiles. finding
- ★ Overlap of variants between eQTL types within a tissue and between liver and muscle within an eQTL type was greater than expected by chance. finding
- Combining geQTL, eeQTL and sQTL increases the chance of identifying loci that regulate gene expression. mechanism
- Quantifying RNA-splicing via intron excision ratios (LeafCutter) provides an accurate splicing phenotype from short reads. method
- ★ Specific genes (FAM184B, CAST, C6) linking eQTL to meat traits are associated with body composition or fatty acid profiles. mechanism
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| bulk RNA-seq (gene & exon expression, intron excision/splicing quantification) | sheep (149 crossbred wether lambs), liver and longissimus dorsi muscle | none (observational, natural genetic variation) | gene read counts, exon read counts, intron excision ratios | Illumina HiSeq2000, paired-end 100 cycle; STAR alignment; FeatureCounts; LeafCutter |
| whole-genome SNP genotyping with imputation to sequence | sheep (149 individuals; reference population of 935 animals) | none | imputed whole-genome SNP genotypes (MAF > 0.05, imputation R2 > 0.4) | Ovine HD SNP Beadchip (~500K); Eagle phasing; Minimac3 imputation |
| cis-eQTL association mapping (linear mixed model) | sheep liver and muscle | none | SNP–molecular phenotype associations within ±1 Mb (geQTL, eeQTL, sQTL; FDR < 0.01) | Wombat software |
| heritability estimation of molecular phenotypes (GREML linear mixed model) | sheep liver and muscle | none | heritability (h2) of gene/exon expression and intron excision | ASReml; GRM from ~500K SNP panel |
| RNA quality assessment | sheep liver and muscle tissue | none | RNA integrity number, 28S/18S ratio | Agilent 2100 Bioanalyzer |
| GWAS enrichment analysis | sheep meat/carcass and fatty acid trait GWAS data | none | enrichment of eQTL in GWAS hits for 56 carcass traits and fatty acid profiles | — |
- – Mean heritability of molecular phenotypes ranged 0.67–0.73 in liver and 0.71–0.77 in muscle (though relatively few were significant at P < 0.05). 0.67–0.77
- – Median distance between eQTL and transcription start sites ranged from 68 to 153 kb across the three eQTL types. 68–153 kb
- ▲ eQTL significantly enriched in GWAS hits for 56 carcass traits and fatty acid profiles.
- ▲ Number of common variants across eQTL types within a tissue and across tissues within an eQTL type was significantly larger than expected by chance.
- – Several geQTL in muscle mapped to FAM184B; hundreds of sQTL in liver and muscle mapped to CAST; hundreds of sQTL in liver mapped to C6.
- mean h2 0.67–0.73 (liver), 0.71–0.77 (muscle) (mean heritability of molecular phenotypes)
- other median 68–153 kb (distance from eQTL to TSS across three eQTL types)
- pvalue FDR < 0.01 (significance threshold for cis-eQTL detection)
- pvalue P < 0.05 (enrichment of eQTL in GWAS hits and overlap greater than chance)
- count 56 carcass/fatty acid traits (GWAS traits showing eQTL enrichment)
- count 149 sheep (crossbred wether lambs used for RNA-seq and genotyping)
- count 13,243 genes / 63,872 exons / 91,699 intron excision events (liver); 12,989 / 60,230 / 87,257 (muscle) (features used to estimate heritability)
- other imputation accuracy 0.97 (average empirical imputation accuracy across target breeds)
Statistical methods review
Model: opusA 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 used RNA-seq from liver and muscle of 149 sheep together with imputed whole-genome SNPs to estimate heritability of molecular phenotypes (gene expression, exon expression, intron excision ratios) and to map cis-eQTL. Heritabilities were estimated with a genomic-relationship linear mixed model in ASReml after adjusting phenotypes for fixed effects via lm() in R; single-SNP cis associations within ±1 Mb were tested with a linear mixed model (polygenic random effect) in Wombat, and significant eQTL were declared at FDR < 0.01. Differences in heritability between phenotype types were compared with the Wilcoxon test, and overlap/enrichment results were assessed against a chance expectation (P < 0.05).
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Linear mixed model (animal/GRM model) for SNP-based heritability | Heritability of gene, exon and intron-excision phenotypes in liver and muscle | 149 individuals | stated |
| Wilcoxon test (wilcox.test in R) | Differences in heritability between the three molecular phenotype types within a tissue | — | not stated |
| Single-SNP linear mixed model (Wombat) with polygenic random effect | Cis-eQTL association of each molecular phenotype with SNPs within ±1 Mb (geQTL, eeQTL, sQTL) | 149 individuals | stated |
| Linear model (lm in R) for fixed-effect adjustment | Pre-adjustment of molecular phenotypes for slaughter day, pen/replicate, dam/sire breed, birth type, dam body condition score | — | not stated |
| Enrichment / overlap test versus chance expectation | Common variants between eQTL types and between tissues, and enrichment of eQTL in GWAS hits for 56 carcass and fatty-acid traits | — | not stated |
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Significant cis-eQTL were declared using a false discovery rate threshold (FDR < 0.01), commonly the Benjamini-Hochberg approach.↳ Could also: A permutation-based per-feature procedure (as implemented in tools like FastQTL/QTLtools or Matrix eQTL with permutations) could also be used to obtain empirical adjusted p-values. — Permutation approaches account for the number and correlation structure of SNPs tested per molecular feature, which can complement a global FDR when many correlated cis-SNPs are evaluated.
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Differences in heritability between molecular phenotype types were compared with the Wilcoxon test.↳ Could also: A Kruskal-Wallis test (for the three types jointly) followed by pairwise comparisons, or a bootstrap on the heritability distributions, could also be used. — A single omnibus test across all three phenotype types provides one family-wise framework for the joint comparison and naturally accommodates more than two groups.
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Heritability point estimates were reported and described by their range across tissues and phenotypes.↳ Could also: Standard errors or 95% confidence intervals from the mixed-model variance estimates could also be reported alongside each estimate. — Interval estimates convey the precision of variance-component estimates, which can be informative given the sample of 149 individuals.
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Phenotypes were pre-adjusted for fixed effects with lm() and the residuals carried into the mixed-model association step.↳ Could also: Fitting the fixed effects jointly within the single mixed model used for association could also be done. — A one-step model propagates uncertainty from the fixed-effect adjustment into the association test rather than treating adjusted phenotypes as known.
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Overlap of variants between eQTL types/tissues and enrichment in GWAS hits were assessed relative to a chance expectation at P < 0.05.↳ Could also: Explicit permutation/resampling or hypergeometric tests with multiplicity control across the enrichment family could also be reported. — Stating the resampling scheme and applying a correction across the set of enrichment comparisons would make the family-wise error rate for these tests explicit.
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Cis-eQTL were tested one SNP at a time within ±1 Mb windows.↳ Could also: Joint/conditional multi-SNP modelling or fine-mapping (e.g., conditional analysis or Bayesian fine-mapping) could also be applied within each window. — Joint modelling can help distinguish independent signals from those reflecting linkage disequilibrium among nearby SNPs.
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-33461502
Paper: Yuan et al. 2021, Expression quantitative trait loci in sheep liver and muscle contribute to variations in meat traits. Genet Sel Evol 53:8. DOI 10.1186/s12711-021-00602-9 · PMCID PMC7812657.
Repo (code): https://github.com/YinLiLin/R-CMplot — this is CMplot, a third-party CRAN/GitHub R package for Manhattan/QQ/circular plots. It is not the authors' analysis pipeline; the paper used it only to draw Fig. S3. Per brief rule P16, applying this named tool to the paper's own data is a valid reproduction.
Data availability (from the paper's "Availability of data and materials")
- WGS genotypes for imputation reference panel → European Variant Archive PRJEB31241 ("Sheep genomes v2", 935 animals). PUBLIC. (This is the accession named in our brief — but it is only the imputation reference, not the eQTL study's own samples.)
- Raw RNA-seq reads of the 149 wether lambs → NCBI PRJNA689847. PUBLIC. Confirmed: 298 RNA-Seq runs = 149 animals × {liver, longissimus muscle}.
- Processed expression matrices, HD SNP genotypes of the 149 animals, and the full eQTL summary statistics → "available from the corresponding author on reasonable request." RESTRICTED (on-request).
- Significant eQTL overlapping GWAS regions (subset, with FDR) → shipped as Additional file 7 / Table S3 (8.3 MB tab-txt). PUBLIC.
Pipeline-derived results & in/out of scope
| Reported result | Pipeline | In scope? | Why |
|---|---|---|---|
| % of 1,130 GWAS regions overlapped by eQTL (geQTL/sQTL, liver/muscle) | distinct GWAS region count / 1130, from eQTL–GWAS overlap | IN | recomputable directly from public Table S3 |
| 298 RNA-seq samples = 149×2 tissues | sample design | IN | verifiable from public ENA PRJNA689847 |
| RNA-seq read-pair counts / library stats (Table S2) | trimming + count | IN (partial) | raw read-pairs from ENA vs paper "clean" pairs |
| Circle/Manhattan plot of eQTL (Fig. S3) | CMplot (the repo) | IN (P16) | run the actual repo on public Table S3 eQTL |
| STAR uniquely-mapped rate per library (Table S2) | STAR → Oar_v3.1 | OUT (last-20%) | needs multi-hour genome index + per-sample align; QC only, not the headline result |
| 640,976 geQTL / 376,181 eeQTL / 678,657 sQTL (liver) etc. — full eQTL counts | Wombat assoc. on HD-imputed genotypes | OUT | requires RESTRICTED HD genotypes + processed data (on request) |
| Heritabilities (0.67–0.77) & #heritable phenotypes | Wombat REML | OUT | requires RESTRICTED genotypes + expression matrices |
Headline biological claim (eQTL discovery) is NOT reproducible from public data — the HD genotypes and full eQTL stats are on-request only. We reproduce the publicly-derivable, clearly-specified numbers (GWAS-overlap %, sample structure, read counts) exactly, and run the named code repo (CMplot) on the public eQTL subset. We do not claim completeness.
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