New method for direct circulating tumor DNA methylation nanopore sequencing based on fragment extension by adaptor_West China Medical Journal

Authors：

SONG Jiajia , PENG Liang ,  CHEN Hao

Department of Laboratory Medicine, West China Hospital, Sichuan University; Clinical Laboratory Medicine Research Center, West China Hospital, Sichuan University; Sichuan Clinical Research Center for Laboratory Medicine; Institution of Medical and Engineering Integration for Molecular Diagnosis, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding?author：

CHEN Hao, Email: haochen@wchscu.edu.cn

Keywords：

Circulating tumor DNA; DNA methylation; nanopore sequencing; adapter ligation; bisulfite-free

DOI：

10.7507/1002-0179.202508034

Video：

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Objective To explore a new method of extending circulating tumor DNA (ctDNA) by adapter ligation to adapt it to nanopore sequencing. Methods A RC adapter was designed to extend ctDNA fragments, and reaction conditions including end repair, dA-tailing, and ligation were optimized. A dual-barcode and RC-adapter-based sample splitting workflow was established, leading to the development of a novel nanopore sequencing based method for ctDNA methylation detection, named RCnano. Results Agarose gel electrophoresis and sequencing results showed that the 48 bp adapter was the optimal length for RCnano. Secondary sample splitting based on RC adapter sequences recovered 42% of unclassified reads, equivalent to a 4%-5% increase in total data yield. Compared with standard nanopore sequencing library preparation, the optimized RCnano workflow increased sequencing output by approximately 6-fold. Among four nanopore methylation callers, Nanopolish performed best, with a mean absolute error of 0.021. RCnano showed good correlation with bisulfite amplicon sequencing (r²=0.952) and was able to detect methylation sites at an abundance as low as 0.1%. Conclusions This study demonstrates a novel application of nanopore sequencing for ultrasensitive ctDNA methylation analysis, which could enhance noninvasive cancer diagnostics and real-time epigenetic monitoring.

Citation： SONG Jiajia, PENG Liang, CHEN Hao. New method for direct circulating tumor DNA methylation nanopore sequencing based on fragment extension by adaptor. West China Medical Journal, 2026, 41(5): 773-778. doi: 10.7507/1002-0179.202508034 Copy

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8.	Liu MC, Oxnard GR, Klein EA, et al. Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA. Ann Oncol, 2021, 31(6): 745-759.
9.	Feng H, Jin P, Wu H. Disease prediction by cell-free DNA methylation. Brief Bioinform, 2019, 20(2): 585-597.
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11.	Bartolomucci A, Nobrega M, Ferrier T, et al. Circulating tumor DNA to monitor treatment response in solid tumors and advance precision oncology. NPJ Precis Oncol, 2025, 9(1): 84.
12.	Zhang Junjie, Xie Shuilian, Xu Jingxiang, et al. Cancer biomarkers discovery of methylation modification with direct high-throughput nanopore sequencing. Front Genet, 2021, 26(12): 672804.
13.	Vaisvila R, Ponnaluri VKC, Sun Z, et al. Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA. Genome Res, 2021, 31(7): 1280-1289.
14.	Si HQ, Wang P, Long F, et al. Cancer liquid biopsies by Oxford nanopore technologies sequencing of cell-free DNA: from basic research to clinical applications. Mol Cancer, 2024, 23(1): 265.
15.	Cheng SH, Jiang P, Sun K, et al. Noninvasive prenatal testing by nanopore sequencing of maternal plasma DNA: feasibility assessment. Clin Chem, 2015, 61(10): 1305-1306.
16.	Martignano F, Munagala U, Crucitta S, et al. Nanopore sequencing from liquid biopsy: analysis of copy number variations from cell-free DNA of lung cancer patients. Mol Cancer, 2021, 20(1): 32.
17.	Marcozzi A, Jager M, Elferink M, et al. Accurate detection of circulating tumor DNA using nanopore consensus sequencing. NPJ Genom Med, 2021, 6(1): 106.
18.	Lau BT, Almeda A, Schauer M, et al. Single-molecule methylation profiles of cell-free DNA in cancer with nanopore sequencing. Genome Med, 2023, 15(1): 33.
19.	Smith ZD, Meissner A. DNA methylation: roles in mammalian development. Nat Rev Genet, 2013, 14(3): 204-220.
20.	Esteller M. Epigenetics in cancer. N Engl J Med, 2008, 358(11): 1148-1159.
21.	Baylin SB, Jones PA. A decade of exploring the cancer epigenome - biological and translational implications. Nat Rev Cancer, 2011, 11(10): 726-734.
22.	Dor Yuval, Cedar Howard. Principles of DNA methylation and their implications for biology and medicine. Lancet, 2018, 392(10149): 777-786.

1. Thierry AR, El Messaoudi S, Gahan PB, et al. Origins, structures, and functions of circulating DNA in oncology. Cancer Metastasis Rev, 2016, 35(3): 347-376.
2. Lo YM, Chan KC, Sun H, et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med, 2010, 2(61): 61ra91.
3. Fiala C, Diamandis EP. Utility of circulating tumor DNA in cancer diagnostics with emphasis on early detection. BMC Med, 2018, 16(1): 166.
4. Ma Liwei, Guo Huiling, Zhao Yunxiang, et al. Liquid biopsy in cancer: current status, challenges and future prospects. Signal Transduct Target Ther, 2024, 9(1): 336.
5. Esteller M, Dawson MA, Kadoch C, et al. The epigenetic hallmarks of cancer. Cancer Discov, 2024, 14(10): 1783-1809.
6. Hai L, Li L, Liu Z, et al. Whole-genome circulating tumor DNA methylation landscape reveals sensitive biomarkers of breast cancer. MedComm, 2022, 3(3): e134.
7. Chen X, Gole J, Gore A, et al. Non-invasive early detection of cancer four years before conventional diagnosis using a blood test. Nat Commun, 2020, 11(1): 3475.
8. Liu MC, Oxnard GR, Klein EA, et al. Sensitive and specific multi-cancer detection and localization using methylation signatures in cell-free DNA. Ann Oncol, 2021, 31(6): 745-759.
9. Feng H, Jin P, Wu H. Disease prediction by cell-free DNA methylation. Brief Bioinform, 2019, 20(2): 585-597.
10. Li L, Sun Y. Circulating tumor DNA methylation detection as biomarker and its application in tumor liquid biopsy: advances and challenges. MedComm (2020), 2024, 5(11): e766.
11. Bartolomucci A, Nobrega M, Ferrier T, et al. Circulating tumor DNA to monitor treatment response in solid tumors and advance precision oncology. NPJ Precis Oncol, 2025, 9(1): 84.
12. Zhang Junjie, Xie Shuilian, Xu Jingxiang, et al. Cancer biomarkers discovery of methylation modification with direct high-throughput nanopore sequencing. Front Genet, 2021, 26(12): 672804.
13. Vaisvila R, Ponnaluri VKC, Sun Z, et al. Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA. Genome Res, 2021, 31(7): 1280-1289.
14. Si HQ, Wang P, Long F, et al. Cancer liquid biopsies by Oxford nanopore technologies sequencing of cell-free DNA: from basic research to clinical applications. Mol Cancer, 2024, 23(1): 265.
15. Cheng SH, Jiang P, Sun K, et al. Noninvasive prenatal testing by nanopore sequencing of maternal plasma DNA: feasibility assessment. Clin Chem, 2015, 61(10): 1305-1306.
16. Martignano F, Munagala U, Crucitta S, et al. Nanopore sequencing from liquid biopsy: analysis of copy number variations from cell-free DNA of lung cancer patients. Mol Cancer, 2021, 20(1): 32.
17. Marcozzi A, Jager M, Elferink M, et al. Accurate detection of circulating tumor DNA using nanopore consensus sequencing. NPJ Genom Med, 2021, 6(1): 106.
18. Lau BT, Almeda A, Schauer M, et al. Single-molecule methylation profiles of cell-free DNA in cancer with nanopore sequencing. Genome Med, 2023, 15(1): 33.
19. Smith ZD, Meissner A. DNA methylation: roles in mammalian development. Nat Rev Genet, 2013, 14(3): 204-220.
20. Esteller M. Epigenetics in cancer. N Engl J Med, 2008, 358(11): 1148-1159.
21. Baylin SB, Jones PA. A decade of exploring the cancer epigenome - biological and translational implications. Nat Rev Cancer, 2011, 11(10): 726-734.
22. Dor Yuval, Cedar Howard. Principles of DNA methylation and their implications for biology and medicine. Lancet, 2018, 392(10149): 777-786.

West China Medical Journal

New method for direct circulating tumor DNA methylation nanopore sequencing based on fragment extension by adaptor

Abstract Full text Figures/Tables Video References Cited by

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