Toward real-world sequencing by microdevice electrophoresis.
Toward real-world sequencing by microdevice electrophoresis.
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通过微型器件电泳实现真实世界的测序。
DOI：
10.1101/gr.9.9.853
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发表时间：
1999
期刊：
影响因子：
7
通讯作者：
Ehrlich,D
中科院分区：
文献类型：
--
作者：
Schmalzing,D;Tsao,N;Koutny,L;Chisholm,D;Srivastava,A;Adourian,A;Linton,L;McEwan,P;Matsudaira,P;Ehrlich,D
We report results using a microdevice for DNA sequencing using samples from chromosome 17, obtained from the Whitehead Institute Center for Genome Research (WICGR) production line. The device had an effective separation distance of 11.5 cm and a lithographically defined injection width of 150 μm. The four-color raw data were processed, base-called by the sequencing software Trout, and compared to the corresponding ABI 377 sequence from WICGR. With a criteria of 99% accuracy, we achieved average continuous reads of 505 bases in 27 min with 3% linear polyacrylamide (LPA) at 150 V/cm, and 460 bases in 22 min with 4% LPA at 200 V/cm at a temperature of 45°C. In the best case, up to 565 bases could be base-called with the same accuracy in <25 min. In some instances, Trout allowed for accurate base-calling down to a resolutionRas low asR= 0.35. This may be due in part to the high signal-to-noise ratio of the microdevice. Unlike many results reported on capillary machines, no additional sample cleanup other than ethanol precipitation was required. In addition, DNA fragment biasing (i.e., discrimination against larger fragments) was reduced significantly through the unique sample injection mechanism of the microfabricated device. This led to increased signal strength for long fragments, which is of great importance for the high performance of the microdevice.
