title: Genome sequencing in microfabricated high-density picolitre reactors.
abstract: The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.
authors.affiliation: 454 Life Sciences Corp., 20 Commercial Street, Branford, Connecticut 06405, USA.
authors.forename: Marcel
authors.initials: M
authors.lastname: Margulies
authors.forename: Michael
authors.initials: M
authors.lastname: Egholm
authors.forename: William E
authors.initials: WE
authors.lastname: Altman
authors.forename: Said
authors.initials: S
authors.lastname: Attiya
authors.forename: Joel S
authors.initials: JS
authors.lastname: Bader
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Bemben
authors.forename: Jan
authors.initials: J
authors.lastname: Berka
authors.forename: Michael S
authors.initials: MS
authors.lastname: Braverman
authors.forename: Yi-Ju
authors.initials: YJ
authors.lastname: Chen
authors.forename: Zhoutao
authors.initials: Z
authors.lastname: Chen
authors.forename: Scott B
authors.initials: SB
authors.lastname: Dewell
authors.forename: Lei
authors.initials: L
authors.lastname: Du
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Fierro
authors.forename: Xavier V
authors.initials: XV
authors.lastname: Gomes
authors.forename: Brian C
authors.initials: BC
authors.lastname: Godwin
authors.forename: Wen
authors.initials: W
authors.lastname: He
authors.forename: Scott
authors.initials: S
authors.lastname: Helgesen
authors.forename: Chun Heen
authors.initials: CH
authors.lastname: Ho
authors.forename: Chun He
authors.initials: CH
authors.lastname: Ho
authors.forename: Gerard P
authors.initials: GP
authors.lastname: Irzyk
authors.forename: Szilveszter C
authors.initials: SC
authors.lastname: Jando
authors.forename: Maria L I
authors.initials: ML
authors.lastname: Alenquer
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Jarvie
authors.forename: Kshama B
authors.initials: KB
authors.lastname: Jirage
authors.forename: Jong-Bum
authors.initials: JB
authors.lastname: Kim
authors.forename: James R
authors.initials: JR
authors.lastname: Knight
authors.forename: Janna R
authors.initials: JR
authors.lastname: Lanza
authors.forename: John H
authors.initials: JH
authors.lastname: Leamon
authors.forename: Steven M
authors.initials: SM
authors.lastname: Lefkowitz
authors.forename: Ming
authors.initials: M
authors.lastname: Lei
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Kenton L
authors.initials: KL
authors.lastname: Lohman
authors.forename: Hong
authors.initials: H
authors.lastname: Lu
authors.forename: Vinod B
authors.initials: VB
authors.lastname: Makhijani
authors.forename: Keith E
authors.initials: KE
authors.lastname: McDade
authors.forename: Michael P
authors.initials: MP
authors.lastname: McKenna
authors.forename: Eugene W
authors.initials: EW
authors.lastname: Myers
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Nickerson
authors.forename: John R
authors.initials: JR
authors.lastname: Nobile
authors.forename: Ramona
authors.initials: R
authors.lastname: Plant
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Puc
authors.forename: Michael T
authors.initials: MT
authors.lastname: Ronan
authors.forename: George T
authors.initials: GT
authors.lastname: Roth
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Sarkis
authors.forename: Jan Fredrik
authors.initials: JF
authors.lastname: Simons
authors.forename: John W
authors.initials: JW
authors.lastname: Simpson
authors.forename: Maithreyan
authors.initials: M
authors.lastname: Srinivasan
authors.forename: Karrie R
authors.initials: KR
authors.lastname: Tartaro
authors.forename: Alexander
authors.initials: A
authors.lastname: Tomasz
authors.forename: Kari A
authors.initials: KA
authors.lastname: Vogt
authors.forename: Greg A
authors.initials: GA
authors.lastname: Volkmer
authors.forename: Shally H
authors.initials: SH
authors.lastname: Wang
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.forename: Michael P
authors.initials: MP
authors.lastname: Weiner
authors.forename: Pengguang
authors.initials: P
authors.lastname: Yu
authors.forename: Richard F
authors.initials: RF
authors.lastname: Begley
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Rothberg
mesh_terms: D019075:Electrophoresis, Capillary; D004655:Emulsions; D005336:Fiber Optic Technology; D016680:Genome, Bacterial; D023281:Genomics / Q000191:economics / Q000295:instrumentation*; D008832:Microchemistry / Q000191:economics / Q000295:instrumentation*; D045704:Mycoplasma genitalium / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000191:economics / Q000295:instrumentation*; D013997:Time Factors
chemical_list: D004655:Emulsions