{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T17:41:29Z","timestamp":1792777289772,"version":"4.5.0"},"reference-count":73,"publisher":"National Academy of Sciences","issue":"43","license":[{"start":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T00:00:00Z","timestamp":1792713600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000196","name":"Canada Foundation for Innovation","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/501100000196","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003579","name":"University of Toronto","doi-asserted-by":"publisher","award":["McLaughlin Centre"],"award-info":[{"award-number":["McLaughlin Centre"]}],"id":[{"id":"10.13039\/501100003579","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008762","name":"Genome Canada","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/100008762","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000165","name":"Sick Kids Foundation","doi-asserted-by":"publisher","award":["Lau Family Endowment in Genome Sciences"],"award-info":[{"award-number":["Lau Family Endowment in Genome Sciences"]}],"id":[{"id":"10.13039\/501100000165","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2026,10,27]]},"abstract":"<jats:p>\n                    The first draft diploid genome assembly (HuRef) of an individual in 2007 was a milestone in genomics. Here, we report HuRef2.0, a revision of HuRef assembled using only Oxford Nanopore Technologies (ONT) Simplex-reads and the hifiasm assembler. Results are close in continuity to the telomere-to-telomere (T2T) assemblies but were assembled from standard DNA samples without using multiple sequencing and mapping technologies, including ultra-long-reads and\/or proximity-ligation. Three ONT flowcells (~103\u00d7 coverage) from fresh blood DNA produced an assembly comprising 26 contigs, with gapless assembly of 23 chromosomes (Chr). Two gaps on ChrY were locally assembled to yield the final T2T-assembly, HuRef2.0, with base accuracy &gt;Q60.34 and 2,393 phase blocks with an NG50 value of 2.36 Mb. Assembly from a single ONT flowcell (~35\u00d7 coverage) consistently produced an assembly more contiguous than GRCh38.p14, providing a foundation for further optimization and scaling. Assembly quality was assessed by direct chromosome-level alignments to a reference genome, variant calling, and annotation of gene-rich regions at Chr22q11, the extended\n                    <jats:italic toggle=\"yes\">MHC<\/jats:italic>\n                    locus, and at several difficult to assemble regions, including the\n                    <jats:italic toggle=\"yes\">rRNA<\/jats:italic>\n                    gene clusters, the subtelomeric region on Chr4q35, ChrY, and the centromeres. More accurate but shorter Pacific Biosciences HiFi-reads produced less contiguous assemblies than from equivalent coverage of error-corrected ONT reads, indicating the importance of read-length. We compared HuRef2.0 to an assembly of a low-passage EBV-transformed lymphoblastoid cell line derived from the same donor. At least at a 400 kb resolution, we did not observe notable structural differences in the two assemblies.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2616836123","type":"journal-article","created":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T17:32:01Z","timestamp":1792776721000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":0,"title":["The revised diploid genome sequence of an individual human: A workflow for near telomere-to-telomere assemblies using only Oxford Nanopore Technologies Simplex-reads"],"prefix":"10.1073","volume":"123","author":[{"given":"Si","family":"Lok","sequence":"first","affiliation":[{"name":"The Centre for Applied Genomics, The Hospital for Sick Children","place":["Toronto, Canada"]},{"name":"Program in Genetics and Genome Biology, The Hospital for Sick Children","place":["Toronto, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Timothy N. H.","family":"Lau","sequence":"additional","affiliation":[{"name":"The Centre for Applied Genomics, The Hospital for Sick Children","place":["Toronto, Canada"]},{"name":"Program in Genetics and Genome Biology, The Hospital for Sick Children","place":["Toronto, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy H. 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S.","family":"Lau","sequence":"additional","affiliation":[{"name":"Division of Genome Diagnostics, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children","place":["Toronto, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian R.","family":"Marshall","sequence":"additional","affiliation":[{"name":"The Centre for Applied Genomics, The Hospital for Sick Children","place":["Toronto, Canada"]},{"name":"Division of Genome Diagnostics, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children","place":["Toronto, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J.","family":"Craig Venter","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/049r1ts75","id-type":"ROR","asserted-by":"publisher"}],"name":"J. Craig Venter Institute","place":["La Jolla"]},{"id":[{"id":"https:\/\/ror.org\/049r1ts75","id-type":"ROR","asserted-by":"publisher"}],"name":"J. Craig Venter Institute","place":["Rockville"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8326-1999","authenticated-orcid":false,"given":"Stephen W.","family":"Scherer","sequence":"additional","affiliation":[{"name":"The Centre for Applied Genomics, The Hospital for Sick Children","place":["Toronto, Canada"]},{"name":"Program in Genetics and Genome Biology, The Hospital for Sick Children","place":["Toronto, Canada"]},{"id":[{"id":"https:\/\/ror.org\/03dbr7087","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular Genetics, Faculty of Medicine, University of Toronto","place":["Toronto, Canada"]},{"id":[{"id":"https:\/\/ror.org\/03dbr7087","id-type":"ROR","asserted-by":"publisher"}],"name":"McLaughlin Centre, University of Toronto","place":["Toronto, Canada"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2026,10,23]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature09792"},{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.1186\/gm483"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/526029a"},{"key":"e_1_3_4_4_2","volume-title":"Economic Impact of the Human Genome Project","author":"Trip S.","year":"2011","unstructured":"S. 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Gao A global view of human centromere variation and evolution. Nature 10.1038\/s41586-026-10841-9 (2026).","DOI":"10.64898\/2025.12.09.693231"},{"key":"e_1_3_4_25_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-47349-7"},{"key":"e_1_3_4_26_2","doi-asserted-by":"crossref","unstructured":"D. Stanojevi\u0107 D. Lin S. Nurk P. F. de Sessions M. \u0160iki\u0107 Telomere-to-telomere phased genome assembly using HERRO-corrected Simplex Nanopore reads. Nature 655 158\u2013165 (2026).","DOI":"10.1038\/s41586-026-10563-y"},{"key":"e_1_3_4_27_2","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-024-07376-y"},{"key":"e_1_3_4_28_2","volume-title":"GATK, and WDL in Terra","author":"O\u2019Gonnor G. A. V. de A.","year":"2020","unstructured":"G. A. V. de A. O\u2019Gonnor, D. Brian, \u201cGenomics in the cloud: Using Docker\u201d in GATK, and WDL in Terra (O\u2019Reilly Media, ed. 1, 2020).","edition":"1"},{"key":"e_1_3_4_29_2","unstructured":"S. Lok Homo sapiens isolate JCVblood-2023-10-26 whole genome shotgun sequencing project. GenBank NCBI. https:\/\/www.ncbi.nlm.nih.gov\/nuccore\/JBPPLP000000000.1. Deposited 31 December 2025."},{"key":"e_1_3_4_30_2","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-020-02134-9"},{"key":"e_1_3_4_31_2","unstructured":"S. Lok Homo sapiens isolate JCVblood-2023-10-26 whole genome shotgun sequencing project. GenBank NCBI. https:\/\/www.ncbi.nlm.nih.gov\/nuccore\/JBTWEK000000000.1. Deposited 08 July 2025."},{"key":"e_1_3_4_32_2","unstructured":"S. Lok Human genome sequencing and assembly of EBV-transformed cells. BioProject NCBI. https:\/\/www.ncbi.nlm.nih.gov\/bioproject\/PRJNA1266743\/. Deposited 23 September 2025."},{"key":"e_1_3_4_33_2","doi-asserted-by":"publisher","DOI":"10.1101\/gr.092759.109"},{"key":"e_1_3_4_34_2","first-page":"1.23.1","article-title":"mtDNA variation and analysis using MITOMAP and MITOMASTER","volume":"1","author":"Lott M. T.","year":"2013","unstructured":"M. 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Lok Human genome sequencing and telomere-to-telomere assembly from fresh blood DNA. BioProject NCBI. https:\/\/www.ncbi.nlm.nih.gov\/bioproject\/PRJNA1266749\/. Deposited 02 June 2025."},{"key":"e_1_3_4_72_2","unstructured":"S. Lok Human genome sequencing and assembly from pacific biosciences HiFi reads. BioProject NCBI. https:\/\/www.ncbi.nlm.nih.gov\/bioproject\/PRJNA1268575\/. Deposited 08 July 2025."},{"key":"e_1_3_4_73_2","doi-asserted-by":"crossref","unstructured":"S. Lok Supporting data for: The revised diploid genome sequence of an individual human: A workflow for near telomere-to-telomere assemblies using only oxford nanopore simplex-reads. Zenodo. https:\/\/doi.org\/10.5281\/zenodo.19861724. 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