Real-time DNA sequencing from single polymerase molecules.
We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.
Eid
John
J
Pacific Biosciences, 1505 Adams Drive, Menlo Park, CA 94025, USA.
Fehr
Adrian
A
Gray
Jeremy
J
Luong
Khai
K
Lyle
John
J
Otto
Geoff
G
Peluso
Paul
P
Rank
David
D
Baybayan
Primo
P
Bettman
Brad
B
Bibillo
Arkadiusz
A
Bjornson
Keith
K
Chaudhuri
Bidhan
B
Christians
Frederick
F
Cicero
Ronald
R
Clark
Sonya
S
Dalal
Ravindra
R
Dewinter
Alex
A
Dixon
John
J
Foquet
Mathieu
M
Gaertner
Alfred
A
Hardenbol
Paul
P
Heiner
Cheryl
C
Hester
Kevin
K
Holden
David
D
Kearns
Gregory
G
Kong
Xiangxu
X
Kuse
Ronald
R
Lacroix
Yves
Y
Lin
Steven
S
Lundquist
Paul
P
Ma
Congcong
C
Marks
Patrick
P
Maxham
Mark
M
Murphy
Devon
D
Park
Insil
I
Pham
Thang
T
Phillips
Michael
M
Roy
Joy
J
Sebra
Robert
R
Shen
Gene
G
Sorenson
Jon
J
Tomaney
Austin
A
Travers
Kevin
K
Trulson
Mark
M
Vieceli
John
J
Wegener
Jeffrey
J
Wu
Dawn
D
Yang
Alicia
A
Zaccarin
Denis
D
Zhao
Peter
P
Zhong
Frank
F
Korlach
Jonas
J
Turner
Stephen
S
D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA / Q000096:biosynthesis; D004270:DNA, Circular / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D007700:Kinetics; D049329:Nanostructures; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D003854:Deoxyribonucleotides; D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D004247:DNA; D004259:DNA-Directed DNA Polymerase