Harris Timothy D, Buzby Phillip R, Babcock Hazen, Beer Eric, Bowers Jayson, Braslavsky Ido, Causey Marie, Colonell Jennifer, Dimeo James, Efcavitch J William, Giladi Eldar, Gill Jaime, Healy John, Jarosz Mirna, Lapen Dan, Moulton Keith, Quake Stephen R, Steinmann Kathleen, Thayer Edward, Tyurina Anastasia, Ward Rebecca, Weiss Howard, Xie Zheng
Helicos BioSciences Corporation, One Kendall Square, Cambridge, MA 02139, USA.
Science. 2008 Apr 4;320(5872):106-9. doi: 10.1126/science.1150427.
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.
只有当数以千计个体的基因组能够被测序用于比较分析时，人类基因组学的全部潜力才能得以实现。参考序列使得短读长得以应用。我们报告了一种无需扩增即可同时测定超过280,000个单个DNA分子核苷酸序列的方法。DNA聚合酶以逐步方式将标记的核苷酸添加到表面固定的引物 - 模板双链体上，并通过荧光成像监测单个DNA分子的异步生长。实现了超过25个碱基的读长以及接近30的等效phred软件程序质量分数。我们使用这种方法对M13病毒进行测序，平均深度超过150倍且覆盖率达100%；因此，我们以高灵敏度突变检测对M13基因组进行了重测序。这展示了一种用于高通量低成本重测序的策略。
