Article

Nature 456, 53-59 (6 November 2008) | doi:10.1038/nature07517; Received 24 June 2008; Accepted 2 October 2008

Accurate whole human genome sequencing using reversible terminator chemistry

David R. Bentley1, Shankar Balasubramanian2, Harold P. Swerdlow1,8, Geoffrey P. Smith1, John Milton1,8, Clive G. Brown1,8, Kevin P. Hall1, Dirk J. Evers1, Colin L. Barnes1,2, Helen R. Bignell1, Jonathan M. Boutell1, Jason Bryant1, Richard J. Carter1, R. Keira Cheetham1, Anthony J. Cox1, Darren J. Ellis1, Michael R. Flatbush3, Niall A. Gormley1, Sean J. Humphray1, Leslie J. Irving1, Mirian S. Karbelashvili3, Scott M. Kirk3, Heng Li4, Xiaohai Liu1,2, Klaus S. Maisinger1, Lisa J. Murray1, Bojan Obradovic1, Tobias Ost1, Michael L. Parkinson1, Mark R. Pratt3, Isabelle M. J. Rasolonjatovo1, Mark T. Reed3, Roberto Rigatti1, Chiara Rodighiero1, Mark T. Ross1, Andrea Sabot1, Subramanian V. Sankar3, Aylwyn Scally4, Gary P. Schroth3, Mark E. Smith1, Vincent P. Smith1, Anastassia Spiridou1, Peta E. Torrance1, Svilen S. Tzonev3, Eric H. Vermaas3, Klaudia Walter4, Xiaolin Wu1, Lu Zhang3, Mohammed D. Alam3, Carole Anastasi1, Ify C. Aniebo1, David M. D. Bailey1, Iain R. Bancarz1, Saibal Banerjee3, Selena G. Barbour1, Primo A. Baybayan3, Vincent A. Benoit1, Kevin F. Benson1, Claire Bevis1, Phillip J. Black1, Asha Boodhun1, Joe S. Brennan1, John A. Bridgham3, Rob C. Brown1, Andrew A. Brown1, Dale H. Buermann3, Abass A. Bundu1, James C. Burrows3, Nigel P. Carter4, Nestor Castillo3, Maria Chiara E. Catenazzi1, Simon Chang3, R. Neil Cooley1, Natasha R. Crake1, Olubunmi O. Dada1, Konstantinos D. Diakoumakos1, Belen Dominguez-Fernandez1, David J. Earnshaw1,2, Ugonna C. Egbujor1, David W. Elmore3, Sergey S. Etchin3, Mark R. Ewan3, Milan Fedurco5, Louise J. Fraser1, Karin V. Fuentes Fajardo1, W. Scott Furey2, David George3, Kimberley J. Gietzen6, Colin P. Goddard1, George S. Golda3, Philip A. Granieri3, David E. Green1, David L. Gustafson3, Nancy F. Hansen7, Kevin Harnish1, Christian D. Haudenschild3, Narinder I. Heyer1, Matthew M. Hims1, Johnny T. Ho3, Adrian M. Horgan1, Katya Hoschler1, Steve Hurwitz3, Denis V. Ivanov3, Maria Q. Johnson3, Terena James1, T. A. Huw Jones1, Gyoung-Dong Kang1, Tzvetana H. Kerelska3, Alan D. Kersey1, Irina Khrebtukova3, Alex P. Kindwall3, Zoya Kingsbury1, Paula I. Kokko-Gonzales1, Anil Kumar1, Marc A. Laurent6, Cynthia T. Lawley6, Sarah E. Lee1, Xavier Lee3, Arnold K. Liao3, Jennifer A. Loch1, Mitch Lok3, Shujun Luo3, Radhika M. Mammen1, John W. Martin3, Patrick G. McCauley1, Paul McNitt3, Parul Mehta1, Keith W. Moon3, Joe W. Mullens3, Taksina Newington1, Zemin Ning4, Bee Ling Ng4, Sonia M. Novo1, Michael J. O'Neill3, Mark A. Osborne1,2, Andrew Osnowski1, Omead Ostadan3,6, Lambros L. Paraschos3, Lea Pickering1, Andrew C. Pike1, Alger C. Pike3, D. Chris Pinkard3, Daniel P. Pliskin3, Joe Podhasky3, Victor J. Quijano3, Come Raczy1, Vicki H. Rae1, Stephen R. Rawlings1, Ana Chiva Rodriguez1, Phyllida M. Roe1, John Rogers1, Maria C. Rogert Bacigalupo1, Nikolai Romanov1, Anthony Romieu5, Rithy K. Roth3, Natalie J. Rourke1, Silke T. Ruediger1, Eli Rusman3, Raquel M. Sanches-Kuiper1, Martin R. Schenker1, Josefina M. Seoane3, Richard J. Shaw1, Mitch K. Shiver3, Steven W. Short3, Ning L. Sizto3, Johannes P. Sluis3, Melanie A. Smith1, Jean Ernest Sohna Sohna1, Eric J. Spence3, Kim Stevens1, Neil Sutton1, Lukasz Szajkowski1, Carolyn L. Tregidgo1, Gerardo Turcatti5, Stephanie vandeVondele1, Yuli Verhovsky3, Selene M. Virk3, Suzanne Wakelin3, Gregory C. Walcott3, Jingwen Wang1, Graham J. Worsley1, Juying Yan3, Ling Yau3, Mike Zuerlein3, Jane Rogers4,8, James C. Mullikin7, Matthew E. Hurles4, Nick J. McCooke1,8, John S. West3, Frank L. Oaks3, Peter L. Lundberg3, David Klenerman2, Richard Durbin4 & Anthony J. Smith1 for

  1. Illumina Cambridge Ltd. (Formerly Solexa Ltd), Chesterford Research Park, Little Chesterford, Nr Saffron Walden, Essex CB10 1XL, UK.
  2. Department of Chemistry, University of Cambridge, The University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, UK.
  3. Illumina Hayward (Formerly Solexa Inc.), 23851 Industrial Boulevard, Hayward, California 94343, USA.
  4. The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
  5. Manteia Predictive Medicine S.A. Zone Industrielle, Coinsins, CH-1267, Switzerland.
  6. Illumina Inc., Corporate Headquarters, 9883 Towne Centre Drive, San Diego, California 92121, USA.
  7. National Human Genome Research Institute, National Institutes of Health, 41 Center Drive, MSC 2132, 9000 Rockville Pike, Bethesda, Maryland 20892-2132, USA.
  8. †Present addresses: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK (H.P.S.); Oxford Nanopore Technologies, Begbroke Science Park, Sandy Lane, Kidlington OX5 1PF, UK (J.M., C.G.B.); BBSRC Genome Analysis Centre, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK (J.R.); Pronota, NV, VIB Bio-Incubator, Technologiepark 4, B-9052 Zwijnaarde/Ghent, Belgium (N.J.M.).

Correspondence to: Correspondence and requests for materials should be addressed to D.R.B. (Email: dbentley@illumina.com).

This article is distributed under the terms of the Creative Commons Attribution-Non-Commercial-Share Alike licence (http://creativecommons.org/licenses/by-nc-sa/3.0/), which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation, and derivative works must be licensed under the same or similar licence.

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DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400–800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30times average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications.

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