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 Tyurina, Rebecca Ward, Howard Weiss, Zheng Xie

 

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 primertemplate 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 >150× and with 100% coverage; thus, we resequenced the M13 genome with highsensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.