Methylation reference datasets from quartet DNA materials for benchmarking epigenome sequencing
The lack of quantitative methylation reference datasets (ground truth) and cross-laboratory reproducibility assessment hinders clinical translation of epigenome-wide sequencing technologies. Using certified Quartet DNA reference materials, here we generate 108 epigenome-sequencing datasets across three mainstream protocols (whole-genome bisulfite sequencing, enzymatic methyl-seq, and TET-assisted pyridine borane sequencing) with triplicates per sample across laboratories. We observe strand-specific methylation biases across all protocols and libraries. Cross-laboratory reproducibility analyses reveal high quantitative methylation levels agreement (mean Pearson correlation coefficient (PCC) = 0.96) but low detection concordance (mean Jaccard index = 0.36). Using consensus voting, we construct genome-wide quantitative methylation reference datasets serving as ground truth for proficiency testing. Key technical parameters-including mean CpG depth, coverage, and strand consistency-correlate strongly with reference-dependent quality metrics (recall, PCC, and RMSE). Collectively, these resources establish foundational standards for benchmarking emerging epigenomic technologies and analytical pipelines, enabling robust, standardized quality control in research and clinical applications.
Authors
- Bo He (ORCID: https://orcid.org/0000-0003-1471-9772)
- Yuanting Zheng (ORCID: https://orcid.org/0000-0003-4480-8303)
- Xiaorou Guo (ORCID: https://orcid.org/0009-0004-7163-9562)
- Sun Yuan (ORCID: https://orcid.org/0000-0002-5608-8836)
- Qingwang Chen (ORCID: https://orcid.org/0000-0002-7191-5801)
- Wanwan Hou (ORCID: https://orcid.org/0000-0002-9813-4554)
- Leming Shi (ORCID: https://orcid.org/0000-0002-2981-4150)
- Yuanfeng Zhang (ORCID: https://orcid.org/0000-0003-2308-019X)
- Yaqing Liu (ORCID: https://orcid.org/0000-0001-9356-9164)
- Luyao Ren (ORCID: https://orcid.org/0000-0002-4052-1148)
- R.C.W. Ma
- Ying Yu (ORCID: https://orcid.org/0000-0002-4084-908X)
- Fujun Qiu
- Bingsi Li (ORCID: https://orcid.org/0000-0002-2114-3647)
- Xiang Fang (ORCID: https://orcid.org/0000-0002-9170-0293)
- Peng Ni (ORCID: https://orcid.org/0000-0002-0801-7574)
- Yujing Zhang (ORCID: https://orcid.org/0000-0002-8265-2488)
- Zhihong Zhang (ORCID: https://orcid.org/0000-0002-1454-1604)
- Rui Zhang (ORCID: https://orcid.org/0000-0003-4660-2042)
- Jianxin Wang (ORCID: https://orcid.org/0000-0003-1516-0480)
- Jinming Li (ORCID: https://orcid.org/0000-0002-1476-3397)
- Lianhua Dong (ORCID: https://orcid.org/0000-0002-8609-9552)
- Weihong Xu (ORCID: https://orcid.org/0000-0003-1552-3333)
- Shumeng Duan
- Yu Ma (ORCID: https://orcid.org/0009-0008-4395-1240)
Institutions
- Central South University (CN)
- Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
- Peking University (CN)
- Fudan University (CN)
- Peking Union Medical College Hospital (CN)
- Fudan University Shanghai Cancer Center (CN)
- Burning Rock Biotech (China) (CN)
- Beijing Hospital (CN)
- Shanghai Institutes for International Studies (CN)
- National Center for Clinical Laboratories (CN)
- National Institute of Metrology (CN)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-10-16
- DOI
- https://doi.org/10.1038/s41467-025-64250-z
- Citations
- 4
- Primary Topic
- Epigenetics and DNA Methylation
- Type
- article
- Field-Weighted Citation Impact
- 3.22
Funders
- Natural Science Foundation of Shanghai
- National Natural Science Foundation of China
- Central South University
- Fudan University
- State Key Laboratory of Genetic Engineering
- Higher Education Discipline Innovation Project
- National Institute of Metrology, China