A 14-bp motif in the KIT active promoter region is critical for melanin accumulation in yaks, mice, and humans.
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.
- ✓Any deviation was negligible
- 🔴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 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
PARTIAL reproduction. The paper is described well enough to map its pipeline, but its key processed artifacts were NOT deposited: there is no 387-yak WY18 joint-call VCF, and 247 of 387 sample accessions are unstated (only ~140 new yaks / 149 runs are public in PRJNA1069901). So the cohort-wide numbers C1-C3 and the Fst/XP-CLR scan C6 are not reproducible from public data (not attempted, documented). The named public code is PopLDdecay, a generic third-party LD tool; the only public processed genotypes are a DIFFERENT study's bovine-pangenome SNP callset. On «our HPC» («job») we: (R2/C4) CONFIRMED the headline KIT locus on the public WY18 reference — the E2F core motif CCGCGGGAAG is present (minus strand) at exactly Chr6:72,971,725 in a GC/CpG-rich active promoter [within-tol]; (R1) built PopLDdecay at the pinned commit 425f978 and ran it on 326 yaks, but the LD-decay curve is uninterpretable because the public pangenome callset is too sparse (1684 Chr6 SNPs) [partial/methodological]; (R3/C5) as a proxy for the indel's weak LD, found 81.5% of flanking yak SNP pairs have r2<0.8 (mean 0.45) — supportive but indirect since the indel is absent from the SNP-only public set [partial]. Did NOT attempt: cohort variant counts, selection scan, GWAS/PCA/ADMIXTURE, RNA-seq DEGs, or any wet-lab claim. This is a feasibility/honesty result, not a refutation: the biological locus checks out on public data, while the cohort statistics depend on undeposited artifacts.
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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
- 2026-06-23
- Rubric version
- v1.0
- Assessed by
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🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-24no 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: sonnetThe study tests whether a specific genetic variant in the KIT gene promoter is the causal regulatory mutation responsible for reduced KIT expression and loss of melanin accumulation (all-white coat phenotype) in yaks, and whether this variant's effect is conserved in mammals including mice and humans.
- ★ A 14-bp deletion in the KIT gene promoter region is associated with the all-white phenotype in yaks, identified via GWAS finding
- ★ The 14-bp deletion lies within an active KIT promoter region and disrupts binding of transcription factors E2F3 and E2F6, reducing KIT expression mechanism
- ★ CRISPR/Cas9 knock-in mice lacking the yak 14-bp promoter motif show reduced KIT expression and white coat spots, confirming functional causality finding
- ★ CRISPR/Cas9 deletion of the human orthologous 14-bp motif in melanoma cells (A375, A875) reduces melanin accumulation finding
- All-white yaks cluster into two distinct genetic groups (W1 and W2) based on NJ tree, PCA, and admixture analysis finding
- ★ The 14-bp promoter sequence is conserved across 12 mammal species and overlaps histone modification marks (H3K4me1, H3K4me3, H3K27ac) in human melanocytes finding
- SCF-cKIT signaling pathway genes show decreased expression in white-coat yak ear tissue compared to black-coat yaks finding
- The 14-bp deletion evolved together with the SCs6 structural variant, jointly contributing to the all-white phenotype in yaks mechanism
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Whole-genome sequencing / GWAS, FST, XP-CLR | 387 domestic and wild yaks (Bos grunniens, B. mutus) | none (natural coat color variants compared) | genetic variants (SNPs/indels), association signals, selection signatures | — |
| PCR genotyping | 40 re-sampled all-white yaks | none | confirmation of 14-bp deletion presence | PCR |
| Dual-luciferase reporter assay | HEK293T cells | promoter fragment truncation/deletion (WT vs Mut lacking 14-bp) | luciferase activity as measure of promoter activity | pGL3-Basic vector with pRL-TK internal reference |
| Dual-luciferase reporter assay with transcription factor co-transfection | HEK293T cells | co-transfection with E2F3 or E2F6 expression plasmids plus WT or Mut promoter | luciferase activity indicating transcription factor binding | pcDNA3.1 vector |
| RNA-seq transcriptome analysis and Western blot | yak ear tissue (6 black-coat, 6 white-coat) | none (natural coat color comparison) | KIT and SCF-cKIT pathway gene expression (PFKM), protein levels | — |
| CRISPR/Cas9 gene knock-in | C57BL/6J transgenic mice | replacement of mouse KIT promoter with yak WT or 14-bp-deleted promoter | coat color phenotype, KIT mRNA expression | CRISPR/Cas9 |
| CRISPR/Cas9 knockout, melanin assay, immunofluorescence, transcriptome (RNA-seq) | human melanoma cell lines A375 and A875 | 24-bp deletion spanning human 14-bp orthologue (hKIT Pro1) vs controls (Pro2, mock) | melanin content, pigment granule localization, differentially expressed genes | CRISPR/Cas9 lentivirus vector |
- – 14-bp deletion at Chr6:72,971,725 is homozygous absent in 20.0% and heterozygous absent in 80.0% of all-white yaks, present in other yaks 20.0%/80.0%
- ▼ WT promoter construct induced significantly higher luciferase activity than Mut (14-bp deleted) construct p<0.01
- ▲ E2F3 and E2F6 co-transfection stimulated expression more with WT plasmid than with Mut plasmid
- ▼ Homozygous ymKIT Pro14Del/Pro14Del mice showed white spots on abdomen and significantly decreased KIT mRNA versus ymKIT+/+ mice
- ▼ hKIT Pro1/Pro1 human melanoma cells showed significantly reduced melanin content relative to wild-type and control cells
- – Transcriptome comparison of hKIT Pro1/Pro1 vs wild-type A375 cells identified differentially expressed genes 1019 upregulated, 1189 downregulated DEGs
- ▼ KIT and other SCF-cKIT pathway genes showed significantly lower expression (PFKM) in white-coat versus black-coat yak ear tissue
- count 387 yaks total (18 wild, 89 all-white, 280 domestic other-colored) (whole-genome sequencing cohort)
- count 13,099,643 high-quality genomic variants (12,229,673 SNPs, 869,970 indels) (variant calling across yak genomes)
- other average sequencing coverage 13.23x, range 8.56x-20.75x (newly sequenced domestic yaks)
- pvalue p<0.01 (luciferase reporter activity of promoter fragments and WT vs Mut comparison)
- count 20.0% homozygous, 80.0% heterozygous absence of 14-bp deletion in all-white yaks (genotype frequency of 14-bp deletion)
- count 40 re-sampled all-white yaks confirmed by PCR (PCR genotyping validation)
- count 1019 upregulated and 1189 downregulated DEGs (hKIT Pro1/Pro1 vs wild-type A375 transcriptome comparison)
- other 5 biological replicates (dual-luciferase assay replicates (Fig. 3D))
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 combined population-genomic analyses (GWAS, Fst, XP-CLR) on whole-genome sequencing data from 387 yaks to map a coat-color locus, then used reporter-gene (luciferase) assays, RNA-seq/FPKM comparisons, transgenic mouse models, and CRISPR-edited human melanoma cell lines to functionally validate a 14-bp KIT promoter deletion. Group comparisons in the functional experiments were primarily analyzed with two-sided Student's t-tests on small numbers of biological replicates, with exact p-values reported and results summarized as mean ± SD; one figure (3B) used letter-based significance grouping across multiple promoter constructs.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Genome-wide association study (GWAS) on whole-genome SNPs/indels | Identifying loci associated with all-white vs. other yak coat color (Fig. 2A) | 387 yaks (18 wild, 89 all-white, 280 other domestic) | not stated |
| Fixation index (Fst) and cross-population composite likelihood ratio (XP-CLR) | Selective sweep analysis around the chr6 and chr25 regions (Fig. 2A, B) | same 387-yak panel | not stated |
| Two-sided Student's t-test | Luciferase reporter activity for WT vs. Mut promoter constructs and E2F3/E2F6 co-transfection (Fig. 3C, D) | five biological replicates | not stated |
| Two-sided Student's t-test | FPKM expression of SCF-cKIT pathway genes, black vs. white yak ear tissue (Fig. 3E) | 6 black-coat and 6 white-coat yak ear tissue samples | not stated |
| Two-sided Student's t-test | KIT mRNA expression in ymKIT+/+ vs. ymKIT Pro14Del/Pro14Del mouse tissue (Fig. 4D); melanin content across human A375/A875 cell genotypes (Fig. 5B, C) | three biological replicates | not stated |
| Letter-based significance grouping (compact letter display, consistent with an omnibus test plus post-hoc comparisons) | Luciferase activity across successive KIT promoter-fragment constructs (Fig. 3B) | not explicitly stated in main text (see Additional file 3: Table S1) | not stated |
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Multiple pairwise comparisons (e.g., four cell genotypes in Fig. 5B–C, several promoter constructs in Fig. 3B) were each assessed with Student's t-tests without a stated multiple-testing correction.↳ Could also: A one-way ANOVA with a post-hoc test (e.g., Tukey HSD) or an explicit Bonferroni/FDR correction applied across the set of pairwise t-tests — This would jointly control the family-wise error rate when several groups or constructs are compared within the same experiment, which pairwise t-tests considered individually do not.
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Gene-expression and phenotypic comparisons based on small numbers of biological replicates (n = 3–6) were assessed with two-sided Student's t-tests.↳ Could also: A non-parametric test such as the Mann-Whitney U (Wilcoxon rank-sum) test — This avoids relying on an assumption of normally distributed values, which can be difficult to verify with such small sample sizes.
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Results throughout the figures are summarized as mean ± SD from a small number of biological replicates.↳ Could also: Reporting a 95% confidence interval alongside or instead of SD — A CI directly conveys the precision of the estimated group difference, which can be informative when replicate numbers are small.
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GWAS, Fst, and XP-CLR were used together to identify signals associated with the all-white phenotype, in a sample that itself showed sub-structure (all-white yaks clustering into two genetic groups, W1/W2).↳ Could also: A mixed linear model association approach that explicitly models kinship/population structure (e.g., GEMMA, GCTA-fastGWA) — Such methods are commonly used to account for confounding from population sub-structure in GWAS, complementing the Fst/XP-CLR sweep-detection approach already used.
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The transcriptome comparison between hKIT Pro1/Pro1 and hKIT Wt/Wt A375 cells reports counts of up- and down-regulated DEGs without specifying the differential-expression testing method or multiple-testing correction in this excerpt.↳ Could also: A standard RNA-seq differential expression pipeline (e.g., DESeq2 or edgeR) with a Benjamini-Hochberg FDR-adjusted p-value threshold — This is a widely used approach for transcriptome-wide comparisons and explicitly addresses the large multiple-testing burden of testing thousands of genes simultaneously.
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Figure 3B uses letter-based grouping ('lowercase letters indicate significant differences') to summarize pairwise significance among several promoter-fragment constructs.↳ Could also: Explicitly naming the omnibus test (e.g., one-way ANOVA) and post-hoc method (e.g., Tukey or Duncan's test) underlying the letter groupings — This would let readers evaluate the specific statistical model and error-rate control behind the compact-letter-display summary.
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — pmid-40629379
Paper: Liu X, et al. "A 14-bp motif in the KIT active promoter region is critical
for melanin accumulation in yaks, mice, and humans." BMC Biol 2025.
PMCID PMC12239316 · DOI 10.1186/s12915-025-02311-x
Named code (Data/Code availability): PopLDdecay — https://github.com/BGI-shenzhen/PopLDdecay
(generic third-party LD tool; pinned commit 425f978ad2dabf16098a6b4bd24bd53a80bb3351, 2024-08-15)
Raw data: SRA PRJNA1069901 (yak WGS) · PRJNA1281012 (A375 RNA-seq)
Reference genome: "WY18" wild-yak assembly, hosted at http://bovpan.lzu.edu.cn (Liu lab, Lanzhou)
1. The pipeline, as described in Methods
WGS of 140 new domestic yaks (avg 13.23×) combined with 247 previously published yaks → 387 yaks (18 wild, 89 all-white, 280 other-colour domestic). Pipeline:
| step | tool (version) | key params |
|---|---|---|
| trim | Cutadapt 2.8 | quality+adapter |
| align | BWA-MEM | default, to WY18 |
| dedup | Picard 2.9.0 MarkDuplicates | — |
| call | GATK 3.8 HaplotypeCaller → GenotypeGVCFs | GVCF joint |
| hard-filter | GATK SelectVariants + HardFilter | QD<2, FS>60/200, MQ<40, MQRankSum<−12.5, ReadPosRankSum<−8/−20 |
| site-filter | VCFtools 0.1.16 | biallelic, MAF>5%, call-rate>80%, HWE<1e−6 |
| phylogeny | phylip NJ + Figtree 1.4.4 | population SNPs |
| structure | ADMIXTURE 1.23 ; PCA GCTA 1.92.4 ; LD-pruning PopLDdecay | — |
| GWAS | PLINK 1.9b4.6 | logistic, PC1–3 covariates, white=1/other=0 |
| selection | VCFtools Fst + XP-CLR | 50 kb window, 25 kb step, black vs white |
| RNA-seq | Hisat2 2.1.0 + StringTie 2.0.6 + Cufflinks | FPKM, DEGs |
2. Reported pipeline-derived numbers (candidate claims)
| id | reported value | location |
|---|---|---|
| C1 | 13,099,643 high-quality variants total | Results / Methods |
| C2 | 12,229,673 SNPs | Results / Methods |
| C3 | 869,970 indels | Results / Methods |
| C4 | 14-bp deletion in KIT promoter at Chr6:72,971,725 | Results, Fig (KIT) |
| C5 | KIT 14-bp indel shows weak LD, r² < 0.8 with nearby & distant variants | Results |
| C6 | Fst + XP-CLR selective-sweep peaks black-vs-white incl. KIT region | Fig (Manhattan) |
| C7 | A375 cells: 1019 up / 1189 down DEGs | Results (RNA-seq) |
3. In scope vs out of scope (feasibility-screened)
Attempted (public artifacts exist, tractable on «our HPC»)
- R1 — PopLDdecay genome-wide LD decay in yaks. Build the named repo (pinned
commit) and run it on the only public yak genotype set — the bovpan
/variation/SNP/snp_Chr6.vcf.gz, subset to the yak samples, paper filter MAF>5%. Reproduces the class of LD analysis the paper ran ("LD-pruning with PopLDdecay"). Caveat: this public VCF is the companion bovine-pangenome callset (536 multi-species samples on assemblyfix_WYDT04, not the paper's 387-yak WY18 callset, which was never deposited). → grade: partial / methodological. - R2 — KIT locus + 14-bp motif on WY18 (C4). Download WY18.fa, locate KIT on Chr6, inspect the promoter region near 72,971,725 for a 14-bp motif/indel context. Verifies the coordinate/sequence basis of the headline finding.
- R3 (best-effort) — "weak LD near KIT" proxy (C5). Compute pairwise r² among the yak SNPs flanking the KIT region in the public Chr6 VCF as a proxy for C5. Heavy caveat: the 14-bp indel itself is absent from the SNP-only public set, and coordinate frames differ (WY18 vs pangenome) — requires locating KIT by alignment.
NOT attempted — hard 80% / blocked (documented, no compute spent)
- C1/C2/C3 (variant counts). Require the full 387-sample cohort. Blockers: (a) only 149 runs / ~2.2 TB are public in PRJNA1069901 — the 247 "previously published" sample accessions are not stated; (b) custom WY18 reference + full GATK joint-calling = multi-TB download + weeks of compute; (c) the authors' own WY18 VCF was not deposited. → not 1:1 reproducible from public artifacts.
- **C6 (Fst/XP-CLR scan), GWAS, PCA, ADMIXTURE
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.
This is a solid, honest partial reproduction, not a refutation. The headline biological claim (C4) checks out 1:1: the KIT active-promoter E2F core motif CCGCGGGAAG is present on the minus strand at exactly Chr6:72,971,725 on the public WY18 reference. The deviation lies entirely on the data-availability/authors' side — the 387-yak WY18 joint-call VCF was never deposited and 247/387 accessions are unstated, so the cohort counts (13,099,643 variants / 12,229,673 SNPs / 869,970 indels) and the Fst/XP-CLR scan are not derivable from released data and the LD work had to run on a different study's pangenome callset. Where comparison was possible the values matched; there is no fabrication signal, only non-derivability of cohort statistics.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
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Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.