Enhanced cfDNA methylation profiling by detection and correction of jagged ends
Analysis of cell-free DNA (cfDNA) methylation can enable noninvasive disease detection by leveraging tissue-specific epigenetic differences between malignant and nonmalignant cells. However, such analyses can be confounded by the repair of jagged DNA fragment ends during double-stranded sequencing library preparation, which introduces spurious methylation signals into cfDNA molecules. Here we present JEEPERS, a framework to detect and correct jagged-end-induced methylation errors while preserving duplex integrity and native methylation patterns. We showed high fidelity for measuring tumor burden and demonstrated allele-specific methylation differences at recurrent somatic mutations, revealing a link between adjacent CpG methylation and EGFR/TP53 driver and resistance mutations. We further demonstrated enhanced diagnostic performance across liquid biopsy applications, including tumor-type-specific methylation profiles, hemimethylation profiling, epigenetic-clock-based age estimation and cell type deconvolution. By faithfully correcting end-repair errors, JEEPERS provides a versatile framework for integrating mutations and methylation features in cfDNA at high resolution, enabling more precise molecular profiling and disease monitoring.
Authors
- Maximilian Diehn (ORCID: https://orcid.org/0000-0003-2032-0581)
- Shuyu Shi
- Ash A. Alizadeh (ORCID: https://orcid.org/0000-0002-5153-5625)
- Takeshi Sugio (ORCID: https://orcid.org/0000-0002-4270-1943)
- Mohammad Shahrokh Esfahani (ORCID: https://orcid.org/0000-0002-0950-3937)
- Feng Tian (ORCID: https://orcid.org/0000-0002-2657-5158)
- Kevin J. Liu (ORCID: https://orcid.org/0000-0003-3836-3213)
- Emily G. Hamilton (ORCID: https://orcid.org/0000-0001-7955-6244)
- Heather A. Wakelee (ORCID: https://orcid.org/0000-0003-1136-980X)
- Xiaoman Kang
- Rene F. Bonilla (ORCID: https://orcid.org/0009-0000-9366-1331)
- Christopher H. Yoo (ORCID: https://orcid.org/0000-0003-2132-8956)
- Chih Long Liu (ORCID: https://orcid.org/0000-0002-6577-5427)
- Nick A. Phillips (ORCID: https://orcid.org/0000-0001-7624-7139)
- Stefan Alig (ORCID: https://orcid.org/0000-0001-6825-702X)
- Tushar Desai (ORCID: https://orcid.org/0000-0002-8794-5319)
- Nicholas H. Juul (ORCID: https://orcid.org/0000-0002-9081-3210)
- Emily C. Shen (ORCID: https://orcid.org/0000-0002-8222-5144)
- Diego Almanza (ORCID: https://orcid.org/0000-0003-1222-9366)
- Cédric Rossi (ORCID: https://orcid.org/0000-0003-3717-7961)
- Joseph B. Shrager (ORCID: https://orcid.org/0000-0003-0859-0589)
- Viswam S. Nair (ORCID: https://orcid.org/0000-0001-6376-8154)
- Emily Chen (ORCID: https://orcid.org/0000-0002-3669-0846)
- William T. Shi (ORCID: https://orcid.org/0000-0001-5452-2902)
- Mari N. Olsen (ORCID: https://orcid.org/0000-0002-8894-4027)
- Angela Hui
- Rui Wang
Institutions
- Sutter Health (US)
- Fred Hutch Cancer Center (US)
- Stanford Medicine (US)
- Stanford Cancer Institute (US)
- Institute for Stem Cell Biology and Regenerative Medicine (US)
- Stanford University (US)
Publication Details
- Journal
- Nature Methods
- Published
- 2026-10-29
- DOI
- https://doi.org/10.1038/s41592-026-03278-5
- Primary Topic
- Cancer Genomics and Diagnostics
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- U.S. Department of Health and Human Services
- LUNGevity Foundation
- Tobacco-Related Disease Research Program
- Virginia and D.K. Ludwig Fund for Cancer Research
- National Institutes of Health