Accurate multiplex polony sequencing of an evolved bacterial genome
Accurate multiplex polony sequencing of an evolved bacterial genome
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DOI：
10.1126/science.1117389
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发表时间：
2005-09-09
期刊：
影响因子：
56.9
通讯作者：
Church, GM
中科院分区：
文献类型：
--
作者：
Shendure, J;Porreca, GJ;Church, GM
我们描述了一种DNA测序技术，其中通常可用的，廉价的荧光显微镜将其转换为快速的非电泳DNA测序自动化。我们将这项技术应用于大肠杆菌的进化菌株，每百万共识基础少于一个错误。一个无细胞的配对库提供了单个DNA分子，这些分子通过乳液聚合酶链反应并平行于1微米珠放大。将数百万的珠子固定在聚丙烯酰胺凝胶中，并通过连接和四色成像进行自动测序周期。每个基础的成本大约是传统测序的成本大约是九分之一。我们的协议是通过现成的仪器和试剂实施的。
We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.