Wiley Graham, Macmil Simone, Qu Chunmei, Wang Ping, Xing Yanbo, White Doug, Li Jianfeng, White James D, Domingo Alexander, Roe Bruce A
University of Oklahoma, Norman, Oklahoma, USA.
Curr Protoc Hum Genet. 2009 Apr;Chapter 18:Unit18.1. doi: 10.1002/0471142905.hg1801s61.
With the introduction of massively parallel, microminiature-based instrumentation for DNA sequencing, robust, reproducible, optimized methods are needed to prepare the target DNA for analysis using these high-throughput approaches because the cost per instrument run is orders of magnitude more than for typical Sanger dideoxynucleotide sequencing on fluorescence-based capillary systems. The methods provided by the manufacturer for genome sequencing using the 454/Roche GS-20 and GS-FLX instruments are robust. However, in an effort to streamline them for automation, we have incorporated several novel changes and deleted several extraneous steps. As a result of modifying these sample preparation protocols, the number of manual manipulations has also been minimized, and the overall yields have been improved for both shotgun and mixed shotgun/paired-end libraries.
随着用于DNA测序的大规模平行、基于微缩技术的仪器的引入，需要稳健、可重复且经过优化的方法来制备用于使用这些高通量方法进行分析的目标DNA，因为每次仪器运行的成本比基于荧光的毛细管系统上的典型桑格双脱氧核苷酸测序高出几个数量级。制造商提供的使用454/罗氏GS-20和GS-FLX仪器进行基因组测序的方法很稳健。然而，为了使其更便于自动化操作，我们引入了一些新颖的改变并删除了几个不必要的步骤。通过修改这些样品制备方案，手动操作的次数也已减至最少，并且鸟枪法文库和混合鸟枪法/双末端文库的总体产量均有所提高。
