Nanopore sequencing and assembly of a human genome with ultra-long reads

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ~30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ~3 Mb). We developed a protocol to generate ultra-long reads (N50 > 100 kb, read lengths up to 882 kb). Incorporating an additional 5× coverage of these ultra-long reads more than doubled the assembly contiguity (NG50 ~6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4-Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length, and closure of gaps in the reference human genome assembly GRCh38.

Related collections

Most cited references 27

Record: found
Abstract: found
Article: not found

An integrated semiconductor device enabling non-optical genome sequencing.

Jonathan Rothberg, Wolfgang Hinz, Todd M. Rearick … (2011)

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

0 comments Cited 549 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Is Open Access

The complete genome of an individual by massively parallel DNA sequencing.

David Wheeler, Maithreyan Srinivasan, Michael Egholm … (2008)

The association of genetic variation with disease and drug response, and improvements in nucleic acid technologies, have given great optimism for the impact of 'genomic medicine'. However, the formidable size of the diploid human genome, approximately 6 gigabases, has prevented the routine application of sequencing methods to deciphering complete individual human genomes. To realize the full potential of genomics for human health, this limitation must be overcome. Here we report the DNA sequence of a diploid genome of a single individual, James D. Watson, sequenced to 7.4-fold redundancy in two months using massively parallel sequencing in picolitre-size reaction vessels. This sequence was completed in two months at approximately one-hundredth of the cost of traditional capillary electrophoresis methods. Comparison of the sequence to the reference genome led to the identification of 3.3 million single nucleotide polymorphisms, of which 10,654 cause amino-acid substitution within the coding sequence. In addition, we accurately identified small-scale (2-40,000 base pair (bp)) insertion and deletion polymorphism as well as copy number variation resulting in the large-scale gain and loss of chromosomal segments ranging from 26,000 to 1.5 million base pairs. Overall, these results agree well with recent results of sequencing of a single individual by traditional methods. However, in addition to being faster and significantly less expensive, this sequencing technology avoids the arbitrary loss of genomic sequences inherent in random shotgun sequencing by bacterial cloning because it amplifies DNA in a cell-free system. As a result, we further demonstrate the acquisition of novel human sequence, including novel genes not previously identified by traditional genomic sequencing. This is the first genome sequenced by next-generation technologies. Therefore it is a pilot for the future challenges of 'personalized genome sequencing'.

0 comments Cited 505 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: found

Is Open Access

Extensive sequencing of seven human genomes to characterize benchmark reference materials

Justin Zook, David Catoe, Jennifer McDaniel … (2016)

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.

0 comments Cited 305 times – based on 0 reviews      Review now

Bookmark

All references

Author and article information

Journal

Journal ID (nlm-journal-id): 9604648

Journal ID (pubmed-jr-id): 20305

Journal ID (nlm-ta): Nat Biotechnol

Journal ID (iso-abbrev): Nat. Biotechnol.

Title: Nature biotechnology

ISSN (Print): 1087-0156

ISSN (Electronic): 1546-1696

Publication date Nihms-submitted: 28 April 2018

Publication date (Electronic): 29 January 2018

Publication date (Print): April 2018

Publication date PMC-release: 04 May 2018

Volume: 36

Issue: 4

Pages: 338-345

Affiliations

[1 ]UC Santa Cruz Genomics Institute, University of California, Santa Cruz, California, USA

[2 ]Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, Bethesda, Maryland, USA

[3 ]Institute of Microbiology and Infection, University of Birmingham, Birmingham, UK

[4 ]Department of Human Genetics, University of Utah, Salt Lake City, Utah, USA

[5 ]USTAR Center for Genetic Discovery, University of Utah, Salt Lake City, Utah, USA

[6 ]Michael Smith Laboratories and Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, Canada

[7 ]Surgical Research Laboratory, Institute of Cancer & Genomic Science, University of Birmingham, UK

[8 ]DeepSeq, School of Life Sciences, University of Nottingham, UK

[9 ]Norwich Medical School, University of East Anglia, Norwich, UK

[10 ]Department of Biomedical Informatics, University of Utah, Salt Lake City, Utah, USA

[11 ]Ontario Institute for Cancer Research, Toronto, Canada

[12 ]Department of Computer Science, University of Toronto, Toronto, Canada

Author notes

Correspondence should be addressed to N.J.L. n.j.loman@ 123456bham.ac.uk or M.L. matt.loose@ 123456nottingham.ac.uk

[13]

These authors contributed equally to this work.

Article

Manuscript ID: NIHMS946406

DOI: 10.1038/nbt.4060

PMC ID: 5889714

PubMed ID: 29431738

SO-VID: a4ca6cd9-6490-4c42-bc74-51130278de06

License:

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

This work is licensed under a Creative Commons Attribution 4.0 International licence. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licence, users will need to obtain permission from the licence holder to reproduce the material. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Nanopore sequencing and assembly of a human genome with ultra-long reads

Read this article at

Abstract

Related collections

Genome Engineering using CRISPR

Most cited references 27

An integrated semiconductor device enabling non-optical genome sequencing.

The complete genome of an individual by massively parallel DNA sequencing.

Extensive sequencing of seven human genomes to characterize benchmark reference materials

Author and article information

Journal

Affiliations

Author notes

Article

History

Categories

Comments

Comment on this article

Similar content 649

Cited by 699

Most referenced authors 2,120