Genome sequencing in microfabricated high-density picolitre reactors.
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.
Margulies
Marcel
M
454 Life Sciences Corp., 20 Commercial Street, Branford, Connecticut 06405, USA.
Egholm
Michael
M
Altman
William E
WE
Attiya
Said
S
Bader
Joel S
JS
Bemben
Lisa A
LA
Berka
Jan
J
Braverman
Michael S
MS
Chen
Yi-Ju
YJ
Chen
Zhoutao
Z
Dewell
Scott B
SB
Du
Lei
L
Fierro
Joseph M
JM
Gomes
Xavier V
XV
Godwin
Brian C
BC
He
Wen
W
Helgesen
Scott
S
Ho
Chun Heen
CH
Ho
Chun He
CH
Irzyk
Gerard P
GP
Jando
Szilveszter C
SC
Alenquer
Maria L I
ML
Jarvie
Thomas P
TP
Jirage
Kshama B
KB
Kim
Jong-Bum
JB
Knight
James R
JR
Lanza
Janna R
JR
Leamon
John H
JH
Lefkowitz
Steven M
SM
Lei
Ming
M
Li
Jing
J
Lohman
Kenton L
KL
Lu
Hong
H
Makhijani
Vinod B
VB
McDade
Keith E
KE
McKenna
Michael P
MP
Myers
Eugene W
EW
Nickerson
Elizabeth
E
Nobile
John R
JR
Plant
Ramona
R
Puc
Bernard P
BP
Ronan
Michael T
MT
Roth
George T
GT
Sarkis
Gary J
GJ
Simons
Jan Fredrik
JF
Simpson
John W
JW
Srinivasan
Maithreyan
M
Tartaro
Karrie R
KR
Tomasz
Alexander
A
Vogt
Kari A
KA
Volkmer
Greg A
GA
Wang
Shally H
SH
Wang
Yong
Y
Weiner
Michael P
MP
Yu
Pengguang
P
Begley
Richard F
RF
Rothberg
Jonathan M
JM
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
D004655:Emulsions