The analysis of cell-free DNA (cfDNA) in plasma provides information on pathological processes in the body. Blood cfDNA is in the form of nucleosomes, which maintain their tissue- and cancer-specific epigenetic state. We developed a single-molecule multiparametric assay to comprehensively profile the epigenetics of plasma-isolated nucleosomes (EPINUC), DNA methylation and cancer-specific protein biomarkers. Our system allows for high-resolution detection of six active and repressive histone modifications and their ratios and combinatorial patterns on millions of individual nucleosomes by single-molecule imaging. In addition, our system provides sensitive and quantitative data on plasma proteins, including detection of non-secreted tumor-specific proteins, such as mutant p53. EPINUC analysis of a cohort of 63 colorectal cancer, 10 pancreatic cancer and 33 healthy plasma samples detected cancer with high accuracy and sensitivity, even at early stages. Finally, combining EPINUC with direct single-molecule DNA sequencing revealed the tissue of origin of colorectal, pancreatic, lung and breast tumors. EPINUC provides multilayered information of potential clinical relevance from limited (<1 ml) liquid biopsy material.
This is a preview of subscription content, access via your institution
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
The datasets generated and analyzed during this study are summarized in Supplementary Tables 1, 3 and 4. BED files of plasma-sequenced reads are available at the Zenodo repository at https://doi.org/10.5281/zenodo.6627498. Image analysis pipelines are available at the Zenodo repository at https://doi.org/10.5281/zenodo.6627723. Data from public repositories used in the study (cBioportal database for CRC primary tumor RNA expression) can be found at https://www.cbioportal.org/study/summary?id=coadread_tcga. Source data are provided with this paper.
Wan, J. C. M. et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat. Rev. Cancer 17, 223–238 (2017).
Bronkhorst, A. J., Ungerer, V. & Holdenrieder, S. The emerging role of cell-free DNA as a molecular marker for cancer management. Biomol. Detect. Quantif. 17, 100087 (2019).
Heitzer, E., Haque, I. S., Roberts, C. E. S. & Speicher, M. R. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat. Rev. Genet. 20, 71–88 (2019).
Lo, Y. M. D., Han, D. S. C., Jiang, P. & Chiu, R. W. K. Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies. Science 372, eaaw3616 (2021).
Xu, R. H. et al. Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma. Nat. Mater. 16, 1155–1162 (2017).
Moss, J. et al. Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease. Nat. Commun. 9, 5068 (2018).
Kang, S. et al. CancerLocator: non-invasive cancer diagnosis and tissue-of-origin prediction using methylation profiles of cell-free DNA. Genome Biol. 18, 53 (2017).
Shen, S. Y. et al. Sensitive tumour detection and classification using plasma cell-free DNA methylomes. Nature 563, 579–583 (2018).
Reinberg, D. & Vales, L. D. Chromatin domains rich in inheritance only certain histone posttranslational modifications qualify as being epigenetic. Science 361, 33–34 (2018).
Shema, E., Bernstein, B. E. & Buenrostro, J. D. Single-cell and single-molecule epigenomics to uncover genome regulation at unprecedented resolution. Nat. Genet. 51, 19–25 (2019).
Allis, C. D. & Jenuwein, T. The molecular hallmarks of epigenetic control. Nat. Rev. Genet. 17, 487–500 (2016).
Mancarella, D. & Plass, C. Epigenetic signatures in cancer: proper controls, current challenges and the potential for clinical translation. Genome Med. 13, 23 (2021).
Sadeh, R. et al. ChIP–seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin. Nat. Biotechnol. 39, 586–598 (2021).
Gezer, U. et al. Histone methylation marks on circulating nucleosomes as novel blood-based biomarker in colorectal cancer. Int. J. Mol. Sci. 16, 29654–29662 (2015).
Van den Ackerveken, P. et al. A novel proteomics approach to epigenetic profiling of circulating nucleosomes. Sci. Rep. 11, 7256 (2021).
Snyder, M. W., Kircher, M., Hill, A. J., Daza, R. M. & Shendure, J. Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell 164, 57–68 (2016).
Ulz, P. et al. Inferring expressed genes by whole-genome sequencing of plasma DNA. Nat. Genet. 48, 1273–1278 (2016).
Sun, K. et al. Orientation-aware plasma cell-free DNA fragmentation analysis in open chromatin regions informs tissue of origin. Genome Res. 29, 418–427 (2019).
Ferlay, J. et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 136, E359–E386 (2015).
Hu, Z. et al. Quantitative evidence for early metastatic seeding in colorectal cancer. Nat. Genet. 51, 1113–1122 (2019).
Shema, E. et al. Single-molecule decoding of combinatorially modified nucleosomes. Science 352, 717–721 (2016).
Heintzman, N. D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007).
Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).
Tiernan, J. P. et al. Carcinoembryonic antigen is the preferred biomarker for in vivo colorectal cancer targeting. Br. J. Cancer 108, 662–667 (2013).
Meng, C. et al. TIMP-1 is a novel serum biomarker for the diagnosis of colorectal cancer: a meta-analysis. PLoS ONE 13, e0207039 (2018).
Yu, J. et al. Identification of MST1 as a potential early detection biomarker for colorectal cancer through a proteomic approach. Sci. Rep. 7, 14265 (2017).
Mandal, S. et al. Direct kinetic fingerprinting for high-accuracy single-molecule counting of diverse disease biomarkers. Acc. Chem. Res. 54, 388–402 (2021).
Furth, N. et al. Unified platform for genetic and serological detection of COVID-19 with single-molecule technology. PLoS ONE 16, e0255096 (2021).
Nakayama, M. & Oshima, M. Mutant p53 in colon cancer. J. Mol. Cell. Biol. 11, 267–276 (2019).
Jung, G., Hernández-Illán, E., Moreira, L., Balaguer, F. & Goel, A. Epigenetics of colorectal cancer: biomarker and therapeutic potential. Nat. Rev. Gastroenterol. Hepatol. 17, 111–130 (2020).
Dawson, M. A. The cancer epigenome: concepts, challenges, and therapeutic opportunities. Science 355, 1147–1152 (2017).
Wood, K. H. & Zhou, Z. Emerging molecular and biological functions of MBD2, a reader of DNA methylation. Front. Genet. 7, 93 (2016).
Bettegowda, C. et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci. Transl. Med. 6, 224ra24 (2014).
Brown, R., Curry, E., Magnani, L., Wilhelm-Benartzi, C. S. & Borley, J. Poised epigenetic states and acquired drug resistance in cancer. Nat. Rev. Cancer 14, 747–753 (2014).
Kerachian, M. A. et al. Crosstalk between DNA methylation and gene expression in colorectal cancer, a potential plasma biomarker for tracing this tumor. Sci. Rep. 10, 2813 (2020).
King, W. D. et al. A cross-sectional study of global DNA methylation and risk of colorectal adenoma. BMC Cancer 14, 488 (2014).
Frederiksen, C. et al. Plasma TIMP-1 levels and treatment outcome in patients treated with XELOX for metastatic colorectal cancer. Ann. Oncol. 22, 369–375 (2011).
Garrido-Laguna, I. & Hidalgo, M. Pancreatic cancer: from state-of-the-art treatments to promising novel therapies. Nat. Rev. Clin. Oncol. 12, 319–334 (2015).
Lubotzky, A. et al. Liquid biopsy reveals collateral tissue damage in cancer. JCI Insight 7, e153559 (2022).
Gai, W. et al. Liver- and colon-specific DNA methylation markers in plasma for investigation of colorectal cancers with or without liver metastases. Clin. Chem. 64, 1239–1249 (2018).
Tannapfel, A. & Reinacher-Schick, A. Chemotherapy associated hepatotoxicity in the treatment of advanced colorectal cancer (CRC). Z. Gastroenterol. 46, 435–440 (2008).
Li, W. et al. 5-Hydroxymethylcytosine signatures in circulating cell-free DNA as diagnostic biomarkers for human cancers. Cell Res. 27, 1243–1257 (2017).
Lio, C. W. J., Yuita, H. & Rao, A. Dysregulation of the TET family of epigenetic regulators in lymphoid and myeloid malignancies. Blood 134, 1487–1497 (2019).
Zhang, L. et al. Tet-mediated covalent labelling of 5-methylcytosine for its genome-wide detection and sequencing. Nat. Commun. 4, 1517 (2013).
Song, C. X. et al. 5-Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages. Cell Res. 27, 1231–1242 (2017).
Newman, A. M. et al. Integrated digital error suppression for improved detection of circulating tumor DNA. Nat. Biotechnol. 34, 547–555 (2016).
Lisanti, S. et al. Comparison of methods for quantification of global DNA methylation in human cells and tissues. PLoS ONE 8, 79044 (2013).
Bock, C. et al. Quantitative comparison of DNA methylation assays for biomarker development and clinical applications. Nat. Biotechnol. 34, 726–737 (2016).
Chandradoss, S. D. et al. Surface passivation for single-molecule protein studies. J. Vis. Exp. 2014, 50549 (2014).
Fleischhacker, M. & Schmidt, B. Circulating nucleic acids (CNAs) and cancer—a survey. Biochim. Biophys. Acta 1775, 181–232 (2007).
Harris, T. D. et al. Single-molecule DNA sequencing of a viral genome. Science 320, 106–109 (2008).
Kim, K. L. et al. Systematic detection of m6A-modified transcripts at single-molecule and single-cell resolution. Cell Rep. Methods 1, 100061 (2021).
We thank R. Rozenzweing, O. Fasust, M. Maurer and R. Irwin for their contributions in establishing a protocol for MBD2 labeling. We thank L. Segev for help with writing and integrating the μManager scripts for performing EPINUC–seq. We are grateful for the important comments made by I. Ulitsky while reading the manuscript. E.S. is an incumbent of the Lisa and Jeffrey Aronin Family Career Development chair. This research was supported by grants from the European Research Council (ERC801655 and ERC_PoC_963863), Emerson Collective, The Israeli Science Foundation (1881/19), The Israel Cancer Research Fund: Research Career Development Award, The German-Israeli Foundation for Scientific Research and Development and Minerva. We also obtained generous support from the Swiss Society Institute for Cancer Prevention Research and the Henry Chanoch Krenter Institute for Biomedical Imaging and Genomics.
Yeda Research and Development Co., Ltd., and SeqLL, Inc., have filed a provisional patent application related to aspects of this publication, and E.S., N.E., V.F., A.S., K.A. and D.J. are named inventors. SeqLL, Inc., has a patent application related to this work (US2016/047747), on which E.S. and D.J. are inventors. E.A., A.S. and D.J. own equity in SeqLL, Inc., where D.J. is an officer and director.
Peer review information
Nature Biotechnology thanks Shixin Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Fedyuk, V., Erez, N., Furth, N. et al. Multiplexed, single-molecule, epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics. Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01447-3