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Accurate whole human genome sequencing using reversible terminator chemistry.

ABSTRACT: 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 >30x 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.

SUBMITTER: Bentley DR

PROVIDER: S-EPMC2581791 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Accurate whole human genome sequencing using reversible terminator chemistry.

Bentley David R DR Balasubramanian Shankar S Swerdlow Harold P HP Smith Geoffrey P GP Milton John J Brown Clive G CG Hall Kevin P KP Evers Dirk J DJ Barnes Colin L CL Bignell Helen R HR Boutell Jonathan M JM Bryant Jason J Carter Richard J RJ Keira Cheetham R R Cox Anthony J AJ Ellis Darren J DJ Flatbush Michael R MR Gormley Niall A NA Humphray Sean J SJ Irving Leslie J LJ Karbelashvili Mirian S MS Kirk Scott M SM Li Heng H Liu Xiaohai X Maisinger Klaus S KS Murray Lisa J LJ Obradovic Bojan B Ost Tobias T Parkinson Michael L ML Pratt Mark R MR Rasolonjatovo Isabelle M J IM Reed Mark T MT Rigatti Roberto R Rodighiero Chiara C Ross Mark T MT Sabot Andrea A Sankar Subramanian V SV Scally Aylwyn A Schroth Gary P GP Smith Mark E ME Smith Vincent P VP Spiridou Anastassia A Torrance Peta E PE Tzonev Svilen S SS Vermaas Eric H EH Walter Klaudia K Wu Xiaolin X Zhang Lu L Alam Mohammed D MD Anastasi Carole C Aniebo Ify C IC Bailey David M D DM Bancarz Iain R IR Banerjee Saibal S Barbour Selena G SG Baybayan Primo A PA Benoit Vincent A VA Benson Kevin F KF Bevis Claire C Black Phillip J PJ Boodhun Asha A Brennan Joe S JS Bridgham John A JA Brown Rob C RC Brown Andrew A AA Buermann Dale H DH Bundu Abass A AA Burrows James C JC Carter Nigel P NP Castillo Nestor N Chiara E Catenazzi Maria M Chang Simon S Neil Cooley R R Crake Natasha R NR Dada Olubunmi O OO Diakoumakos Konstantinos D KD Dominguez-Fernandez Belen B Earnshaw David J DJ Egbujor Ugonna C UC Elmore David W DW Etchin Sergey S SS Ewan Mark R MR Fedurco Milan M Fraser Louise J LJ Fuentes Fajardo Karin V KV Scott Furey W W George David D Gietzen Kimberley J KJ Goddard Colin P CP Golda George S GS Granieri Philip A PA Green David E DE Gustafson David L DL Hansen Nancy F NF Harnish Kevin K Haudenschild Christian D CD Heyer Narinder I NI Hims Matthew M MM Ho Johnny T JT Horgan Adrian M AM Hoschler Katya K Hurwitz Steve S Ivanov Denis V DV Johnson Maria Q MQ James Terena T Huw Jones T A TA Kang Gyoung-Dong GD Kerelska Tzvetana H TH Kersey Alan D AD Khrebtukova Irina I Kindwall Alex P AP Kingsbury Zoya Z Kokko-Gonzales Paula I PI Kumar Anil A Laurent Marc A MA Lawley Cynthia T CT Lee Sarah E SE Lee Xavier X Liao Arnold K AK Loch Jennifer A JA Lok Mitch M Luo Shujun S Mammen Radhika M RM Martin John W JW McCauley Patrick G PG McNitt Paul P Mehta Parul P Moon Keith W KW Mullens Joe W JW Newington Taksina T Ning Zemin Z Ling Ng Bee B Novo Sonia M SM O'Neill Michael J MJ Osborne Mark A MA Osnowski Andrew A Ostadan Omead O Paraschos Lambros L LL Pickering Lea L Pike Andrew C AC Pike Alger C AC Chris Pinkard D D Pliskin Daniel P DP Podhasky Joe J Quijano Victor J VJ Raczy Come C Rae Vicki H VH Rawlings Stephen R SR Chiva Rodriguez Ana A Roe Phyllida M PM Rogers John J Rogert Bacigalupo Maria C MC Romanov Nikolai N Romieu Anthony A Roth Rithy K RK Rourke Natalie J NJ Ruediger Silke T ST Rusman Eli E Sanches-Kuiper Raquel M RM Schenker Martin R MR Seoane Josefina M JM Shaw Richard J RJ Shiver Mitch K MK Short Steven W SW Sizto Ning L NL Sluis Johannes P JP Smith Melanie A MA Ernest Sohna Sohna Jean J Spence Eric J EJ Stevens Kim K Sutton Neil N Szajkowski Lukasz L Tregidgo Carolyn L CL Turcatti Gerardo G Vandevondele Stephanie S Verhovsky Yuli Y Virk Selene M SM Wakelin Suzanne S Walcott Gregory C GC Wang Jingwen J Worsley Graham J GJ Yan Juying J Yau Ling L Zuerlein Mike M Rogers Jane J Mullikin James C JC Hurles Matthew E ME McCooke Nick J NJ West John S JS Oaks Frank L FL Lundberg Peter L PL Klenerman David D Durbin Richard R Smith Anthony J AJ

Nature 20081101 7218

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 ...[more]

PMID: 18987734

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Project description:Familial apparently balanced translocations (ABTs) segregating with discordant phenotypes are extremely challenging for interpretation and counseling due to the scarcity of publications and lack of routine techniques for quick investigation. Recently, next generation sequencing has emerged as an efficacious methodology for precise detection of translocation breakpoints. However, studies so far have mainly focused on de novo translocations. The present study focuses specifically on familial cases in order to shed some light to this diagnostic dilemma. Whole-genome mate-pair sequencing (WG-MPS) was applied to map the breakpoints in nine two-way ABT carriers from four families. Translocation breakpoints and patient-specific structural variants were validated by Sanger sequencing and quantitative Real Time PCR, respectively. Identical sequencing patterns and breakpoints were identified in affected and non-affected members carrying the same translocations. PTCD1, ATP5J2-PTCD1, CADPS2, and STPG1 were disrupted by the translocations in three families, rendering them initially as possible disease candidate genes. However, subsequent mutation screening and structural variant analysis did not reveal any pathogenic mutations or unique variants in the affected individuals that could explain the phenotypic differences between carriers of the same translocations. In conclusion, we suggest that NGS-based methods, such as WG-MPS, can be successfully used for detailed mapping of translocation breakpoints, which can also be used in routine clinical investigation of ABT cases. Unlike de novo translocations, no associations were determined here between familial two-way ABTs and the phenotype of the affected members, in which the presence of cryptic imbalances and complex chromosomal rearrangements has been excluded. Future whole-exome or whole-genome sequencing will potentially reveal unidentified mutations in the patients underlying the discordant phenotypes within each family. In addition, larger studies are needed to determine the exact percentage for phenotypic risk in families with ABTs.

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Accurate whole human genome sequencing using reversible terminator chemistry.

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Accurate whole human genome sequencing using reversible terminator chemistry.

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