Single-molecule DNA sequencing of a viral genome

Timothy D. Harris, Phillip R. Buzby, Hazen Babcock, Eric Beer, Jayson Bowers, Ido Braslavsky, Marie Causey, Jennifer Colonell, James DiMeo, J. William Efcavitch, Eldar Giladi, Jaime Gill, John Healy, Mirna Jarosz, Dan Lapen, Keith Moulton, Stephen R. Quake, Kathleen Steinmann, Edward Thayer, Anastasia TyurinaRebecca Ward, Howard Weiss, Zheng Xie

Research output: Contribution to journalArticlepeer-review

540 Scopus citations

Abstract

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.

Original languageAmerican English
Pages (from-to)106-109
Number of pages4
JournalScience
Volume320
Issue number5872
DOIs
StatePublished - 5 Apr 2008
Externally publishedYes

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