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Highly-accurate long-read sequencing improves variant detection and assembly of a human genome

View ORCID ProfileAaron M. Wenger, Paul Peluso, View ORCID ProfileWilliam J. Rowell, Pi-Chuan Chang, Richard J. Hall, Gregory T. Concepcion, Jana Ebler, Arkarachai Fungtammasan, Alexey Kolesnikov, Nathan D. Olson, Armin Töpfer, Michael Alonge, View ORCID ProfileMedhat Mahmoud, View ORCID ProfileYufeng Qian, View ORCID ProfileChen-Shan Chin, Adam M. Phillippy, Michael C. Schatz, Gene Myers, Mark A. DePristo, View ORCID ProfileJue Ruan, Tobias Marschall, View ORCID ProfileFritz J. Sedlazeck, View ORCID ProfileJustin M. Zook, View ORCID ProfileHeng Li, Sergey Koren, Andrew Carroll, David R. Rank, Michael W. Hunkapiller
doi: https://doi.org/10.1101/519025
Aaron M. Wenger
1Pacific Biosciences, Menlo Park, CA, USA
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Paul Peluso
1Pacific Biosciences, Menlo Park, CA, USA
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William J. Rowell
1Pacific Biosciences, Menlo Park, CA, USA
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Pi-Chuan Chang
2Google Inc., Mountain View, CA, USA
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Richard J. Hall
1Pacific Biosciences, Menlo Park, CA, USA
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Gregory T. Concepcion
1Pacific Biosciences, Menlo Park, CA, USA
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Jana Ebler
3Center for Bioinformatics, Saarland University, Saarbrücken, Germany
4Max Planck Institute for Informatics, Saarland Informatics Campus E1.4, Saarbrücken, Germany
5Graduate School of Computer Science, Saarland University, Saarland Informatics Campus E1.3, Saarbrücken, Germany
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Arkarachai Fungtammasan
6DNAnexus, Mountain View, CA, USA
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Alexey Kolesnikov
2Google Inc., Mountain View, CA, USA
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Nathan D. Olson
7National Institute of Standards and Technology, Gaithersburg, MD, USA
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Armin Töpfer
1Pacific Biosciences, Menlo Park, CA, USA
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Michael Alonge
8Department of Computer Science, Johns Hopkins University, Baltimore, MD, USA
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Medhat Mahmoud
9Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA
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Yufeng Qian
1Pacific Biosciences, Menlo Park, CA, USA
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Chen-Shan Chin
6DNAnexus, Mountain View, CA, USA
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Adam M. Phillippy
10Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, Bethesda, MD, USA
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Michael C. Schatz
8Department of Computer Science, Johns Hopkins University, Baltimore, MD, USA
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Gene Myers
11Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
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Mark A. DePristo
2Google Inc., Mountain View, CA, USA
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Jue Ruan
12Agricultural Genomics Institute, Chinese Academy of Agriculture Sciences, Shenzen, China
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Tobias Marschall
3Center for Bioinformatics, Saarland University, Saarbrücken, Germany
4Max Planck Institute for Informatics, Saarland Informatics Campus E1.4, Saarbrücken, Germany
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Fritz J. Sedlazeck
9Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA
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Justin M. Zook
7National Institute of Standards and Technology, Gaithersburg, MD, USA
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Heng Li
13Dana-Farber Cancer Institute, Boston, MA, USA
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Sergey Koren
10Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, Bethesda, MD, USA
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Andrew Carroll
2Google Inc., Mountain View, CA, USA
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David R. Rank
1Pacific Biosciences, Menlo Park, CA, USA
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  • For correspondence: mhunkapiller@pacb.com drank@pacb.com
Michael W. Hunkapiller
1Pacific Biosciences, Menlo Park, CA, USA
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  • For correspondence: mhunkapiller@pacb.com drank@pacb.com
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Abstract

The major DNA sequencing technologies in use today produce either highly-accurate short reads or noisy long reads. We developed a protocol based on single-molecule, circular consensus sequencing (CCS) to generate highly-accurate (99.8%) long reads averaging 13.5 kb and applied it to sequence the well-characterized human HG002/NA24385. We optimized existing tools to comprehensively detect variants, achieving precision and recall above 99.91% for SNVs, 95.98% for indels, and 95.99% for structural variants. We estimate that 2,434 discordances are correctable mistakes in the high-quality Genome in a Bottle benchmark. Nearly all (99.64%) variants are phased into haplotypes, which further improves variant detection. De novo assembly produces a highly contiguous and accurate genome with contig N50 above 15 Mb and concordance of 99.998%. CCS reads match short reads for small variant detection, while enabling structural variant detection and de novo assembly at similar contiguity and markedly higher concordance than noisy long reads.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted January 23, 2019.
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Highly-accurate long-read sequencing improves variant detection and assembly of a human genome
Aaron M. Wenger, Paul Peluso, William J. Rowell, Pi-Chuan Chang, Richard J. Hall, Gregory T. Concepcion, Jana Ebler, Arkarachai Fungtammasan, Alexey Kolesnikov, Nathan D. Olson, Armin Töpfer, Michael Alonge, Medhat Mahmoud, Yufeng Qian, Chen-Shan Chin, Adam M. Phillippy, Michael C. Schatz, Gene Myers, Mark A. DePristo, Jue Ruan, Tobias Marschall, Fritz J. Sedlazeck, Justin M. Zook, Heng Li, Sergey Koren, Andrew Carroll, David R. Rank, Michael W. Hunkapiller
bioRxiv 519025; doi: https://doi.org/10.1101/519025
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Highly-accurate long-read sequencing improves variant detection and assembly of a human genome
Aaron M. Wenger, Paul Peluso, William J. Rowell, Pi-Chuan Chang, Richard J. Hall, Gregory T. Concepcion, Jana Ebler, Arkarachai Fungtammasan, Alexey Kolesnikov, Nathan D. Olson, Armin Töpfer, Michael Alonge, Medhat Mahmoud, Yufeng Qian, Chen-Shan Chin, Adam M. Phillippy, Michael C. Schatz, Gene Myers, Mark A. DePristo, Jue Ruan, Tobias Marschall, Fritz J. Sedlazeck, Justin M. Zook, Heng Li, Sergey Koren, Andrew Carroll, David R. Rank, Michael W. Hunkapiller
bioRxiv 519025; doi: https://doi.org/10.1101/519025

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