Genome sequencing in microfabricated high-density picolitre reactors
Genome sequencing in microfabricated high-density picolitre reactors
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DOI：
10.1038/nature03959
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发表时间：
2005-09-15
期刊：
影响因子：
64.8
通讯作者：
Rothberg, JM
中科院分区：
文献类型：
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作者：
Margulies, M;Egholm, M;Rothberg, JM
近年来，大规模DNA测序项目的激增推动了对替代方法的研究，以减少时间和成本。在这里，我们描述了一个可扩展的，高度并行的测序系统，原始吞吐量显着大于国家的最先进的毛细管电泳仪器。该仪器使用了一种新颖的光纤载玻片，每个威尔斯孔都有，在一次四小时的运行中，能够以99%或更高的准确度对2500万个碱基进行测序。为了实现超过当前桑格测序技术的约100倍的通量增加，我们开发了用于DNA扩增的乳液方法和使用针对固体支持物和皮升规模体积优化的焦磷酸测序方案的通过合成进行测序的仪器。在这里，我们通过鸟枪测序和生殖支原体基因组的从头组装显示了该系统的实用性、通量、准确性和稳健性，在一次机器运行中具有96%的覆盖率和99.96%的准确性。
The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.