Gapless assembly of complete human and plant chromosomes using only nanopore sequencing

The combination of ultra-long (UL) Oxford Nanopore Technologies (ONT) sequencing reads with long, accurate Pacific Bioscience (PacBio) High Fidelity (HiFi) reads has enabled the completion of a human genome and spurred similar efforts to complete the genomes of many other species. However, this approach for complete, "telomere-to-telomere" genome assembly relies on multiple sequencing platforms, limiting its accessibility. ONT "Duplex" sequencing reads, where both strands of the DNA are read to improve quality, promise high per-base accuracy. To evaluate this new data type, we generated ONT Duplex data for three widely studied genomes: human HG002, <i>Solanum lycopersicum</i> Heinz 1706 (tomato), and <i>Zea mays</i> B73 (maize). For the diploid, heterozygous HG002 genome, we also used "Pore-C" chromatin contact mapping to completely phase the haplotypes. We found the accuracy of Duplex data to be similar to HiFi sequencing, but with read lengths tens of kilobases longer, and the Pore-C data to be compatible with existing diploid assembly algorithms. This combination of read length and accuracy enables the construction of a high-quality initial assembly, which can then be further resolved using the UL reads, and finally phased into chromosome-scale haplotypes with Pore-C. The resulting assemblies have a base accuracy exceeding 99.999% (Q50) and near-perfect continuity, with most chromosomes assembled as single contigs. We conclude that ONT sequencing is a viable alternative to HiFi sequencing for de novo genome assembly, and provides a multirun single-instrument solution for the reconstruction of complete genomes.

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Journal
Genome Research
Published
2024-11-01
DOI
https://doi.org/10.1101/gr.279334.124
Citations
31
Primary Topic
Genomics and Phylogenetic Studies
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article

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article

Gapless assembly of complete human and plant chromosomes using only nanopore sequencing

Koen Nijbroek, Brandy McNulty, Erik Garrison, Dick Roelofs et al.
31 citations
Genome Research
Genomics and Phylogenetic Studies
article

Gapless assembly of complete human and plant chromosomes using only nanopore sequencing

Koen Nijbroek, Brandy McNulty, Erik Garrison, Dick Roelofs, Mikko Rautiainen, Julian Lucas, Andrea Guarracino, Sanwen Huang, Zhigui Bao, Alexander Wittenberg, Sergey Koren, Jimin Park, Sergey Nurk, Michael C. Schatz, Karen H. Miga, Katherine R. Lawrence, Katharine M. Jenike, W. Richard McCombie, Sara Goodwin, Mike Vella, Doreen Ware, Arang Rhie, Shujun Ou, Ilse Vrijenhoek, Adam M. Phillippy, Harrie Schneiders, Olle Nordesjö
article en
31 citations

Abstract

The combination of ultra-long (UL) Oxford Nanopore Technologies (ONT) sequencing reads with long, accurate Pacific Bioscience (PacBio) High Fidelity (HiFi) reads has enabled the completion of a human genome and spurred similar efforts to complete the genomes of many other species. However, this approach for complete, "telomere-to-telomere" genome assembly relies on multiple sequencing platforms, limiting its accessibility. ONT "Duplex" sequencing reads, where both strands of the DNA are read to improve quality, promise high per-base accuracy. To evaluate this new data type, we generated ONT Duplex data for three widely studied genomes: human HG002, <i>Solanum lycopersicum</i> Heinz 1706 (tomato), and <i>Zea mays</i> B73 (maize). For the diploid, heterozygous HG002 genome, we also used "Pore-C" chromatin contact mapping to completely phase the haplotypes. We found the accuracy of Duplex data to be similar to HiFi sequencing, but with read lengths tens of kilobases longer, and the Pore-C data to be compatible with existing diploid assembly algorithms. This combination of read length and accuracy enables the construction of a high-quality initial assembly, which can then be further resolved using the UL reads, and finally phased into chromosome-scale haplotypes with Pore-C. The resulting assemblies have a base accuracy exceeding 99.999% (Q50) and near-perfect continuity, with most chromosomes assembled as single contigs. We conclude that ONT sequencing is a viable alternative to HiFi sequencing for de novo genome assembly, and provides a multirun single-instrument solution for the reconstruction of complete genomes.

Genome ResearchVol. 34(11)
Chinese Academy of Tropical Agricultural Sciences (CN), National Institutes of Health (US), Johns Hopkins University (US), University of Tennessee Health Science Center (US), University of California, Santa Cruz (US), Cold Spring Harbor Laboratory (US), Agricultural Genomics Institute at Shenzhen (CN), National Human Genome Research Institute (US), KeyGene (Netherlands) (NL), Chinese Academy of Agricultural Sciences (CN), Ministry of Agriculture and Rural Affairs (CN), Oxford Nanopore Technologies (United Kingdom) (GB), Max Planck Institute for Biology (DE), The Ohio State University (US), Ithaca College (US)
National Science Foundation, Cold Spring Harbor Laboratory, Northwell Health, Human Frontier Science Program, National Natural Science Foundation of China, Max-Planck-Gesellschaft, National Institutes of Health, National Key Research and Development Program of China, National Human Genome Research Institute
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Genomics and Phylogenetic Studies
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