Single-molecule DNA sequencing of a viral genome.
The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.
Harris
Timothy D
TD
Helicos BioSciences Corporation, One Kendall Square, Cambridge, MA 02139, USA. tharris@helicosbio.com
Buzby
Phillip R
PR
Babcock
Hazen
H
Beer
Eric
E
Bowers
Jayson
J
Braslavsky
Ido
I
Causey
Marie
M
Colonell
Jennifer
J
Dimeo
James
J
Efcavitch
J William
JW
Giladi
Eldar
E
Gill
Jaime
J
Healy
John
J
Jarosz
Mirna
M
Lapen
Dan
D
Moulton
Keith
K
Quake
Stephen R
SR
Steinmann
Kathleen
K
Thayer
Edward
E
Tyurina
Anastasia
A
Ward
Rebecca
R
Weiss
Howard
H
Xie
Zheng
Z
D000465:Algorithms; D017104:Bacteriophage M13 / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D017931:DNA Primers; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics*; D016679:Genome, Viral; D009154:Mutation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D013698:Templates, Genetic
D017931:DNA Primers; D004279:DNA, Viral