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Peer-reviewedCC BY-NC-ND 4.0Proceedings of the National Academy of Sciences of the United States of America

The revised diploid genome sequence of an individual human: A workflow for near telomere-to-telomere assemblies using only Oxford Nanopore Technologies Simplex-reads

  • Si Lok1,2
  • Timothy N. H. Lau1,2
  • Amy H. Y. Tong3
  • Brett Trost1,2,4,5
  • Miriam S. Reuter1,2
  • Bhooma Thiruvahindrapuram1,2
  • Tara A. Paton1,2
  • Jeffrey R. MacDonald1,2
  • Lynette Y. S. Lau6
  • Christian R. Marshall1,6
  • J. Craig Venter7,8
  • Stephen W. Scherer1,2,5,9
9 affiliations
  1. 1The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
  2. 2Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
  3. 3Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada
  4. 4Program in Molecular Medicine, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
  5. 5Department of Molecular Genetics, Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada
  6. 6Division of Genome Diagnostics, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada
  7. 7J. Craig Venter Institute, La Jolla, CA 92037
  8. 8J. Craig Venter Institute, Rockville, MD 20850
  9. 9McLaughlin Centre, University of Toronto, Toronto, ON M5G 0A4, Canada
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Abstract

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× 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 >Q60.34 and 2,393 phase blocks with an NG50 value of 2.36 Mb. Assembly from a single ONT flowcell (~35× 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 MHC locus, and at several difficult to assemble regions, including the rRNA 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.

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Cite

BibTeXRIS
BibTeX
@article{lok2026revised,
  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}},
  author = {Si Lok and Timothy N. H. Lau and Amy H. Y. Tong and Brett Trost and Miriam S. Reuter and Bhooma Thiruvahindrapuram and Tara A. Paton and Jeffrey R. MacDonald and Lynette Y. S. Lau and Christian R. Marshall and J. Craig Venter and Stephen W. Scherer},
  journal = {Proceedings of the National Academy of Sciences of the United States of America},
  year = {2026},
  month = oct,
  doi = {10.1073/pnas.2616836123},
  url = {https://doi.org/10.1073/pnas.2616836123},
  note = {CC BY-NC-ND 4.0},
}
RIS
TY  - JOUR
TI  - The revised diploid genome sequence of an individual human: A workflow for near telomere-to-telomere assemblies using only Oxford Nanopore Technologies Simplex-reads
AU  - Si Lok
AU  - Timothy N. H. Lau
AU  - Amy H. Y. Tong
AU  - Brett Trost
AU  - Miriam S. Reuter
AU  - Bhooma Thiruvahindrapuram
AU  - Tara A. Paton
AU  - Jeffrey R. MacDonald
AU  - Lynette Y. S. Lau
AU  - Christian R. Marshall
AU  - J. Craig Venter
AU  - Stephen W. Scherer
PY  - 2026
DA  - 2026/10/23
JO  - Proceedings of the National Academy of Sciences of the United States of America
DO  - 10.1073/pnas.2616836123
UR  - https://doi.org/10.1073/pnas.2616836123
AB  - 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× 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 >Q60.34 and 2,393 phase blocks with an NG50 value of 2.36 Mb. Assembly from a single ONT flowcell (~35× 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 MHC locus, and at several difficult to assemble regions, including the rRNA 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.
SN  - 0027-8424
SN  - 1091-6490
ER  -