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.
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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.
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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.
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Fedyuk, V., Erez, N., Furth, N. et al. Multiplexed, single-molecule, epigenetic analysis of plasma-isolated nucleosomes for cancer diagnostics. Nat Biotechnol 41, 212–221 (2023). https://doi.org/10.1038/s41587-022-01447-3
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