OKLAHOMA UNIVERSITY GENOME CENTER - ADVANCED CENTER FOR

这项工作的主要目标是继续扩大我们的基因组中心，并对至少5%的小鼠基因组进行测序，至少99%的准确率，为Pieter de Jong博士的小鼠C57BL6/J BAC文库中随机BAC的大约5倍覆盖率的工作草案。高生物学兴趣的BACs由小鼠社区提供，并由约翰霍普金斯大学的Reeves博士验证。这大约150mbp的小鼠基因组序列，其中包括大约30mbp（99.99%的准确度），可以通过a)引物从基于pucc的亚克隆和/或BAC上走下来，b)通过基于多重pcr的测序或c)两者的组合完成，到01年底，大约120mb或工作草图序列（准确度超过99%）。在2002年，将产生额外的约30 Mbp的已完工和约270 Mbp的工作草图序列。在2003年，我们将集中精力完成前两年产生的数据和/或在与NHGRI工作人员和NHGRI咨询委员会协商后生成额外的工作草案数据。大约10%的克隆将进入我们的测序队列，这是由小鼠基因组Oversite委员会推荐的，并由约翰霍普金斯大学的Reeves博士通过分包合同支持验证。我们的中心建立在一个成功的并行结构，研究生/博士后培训为基础的基因组测序方法上，现在已经完成并存储了超过1000万个完成的人类和小鼠基因组DNA序列到GenBank中，总成本不到28美分/个碱基，按GenBank中完成的总碱基数计算。我们的成功部分是基于a)我们富有成效的合作，例如与Reeves博士一起完成了近3 Mb的小鼠基因组DNA序列，b)我们的组织模式导致了改进的培训方法和更有效的测序策略和方法，以及c)我们有效地实施了廉价的引物导向的基于聚合酶链反应的封闭策略。我们计划在第一年将员工人数增加50%，并将测序相关的设备和用品增加一倍，在第二年也会有类似的增长，在第三年则会有较小的增长。为了扩大规模，我们的实验室现在已经重组为4个相互交织的小组，管理，支持，研究和测序，以及15个平行的序列生产小组，准备从人类或细菌基因组测序转化为小鼠基因组测序。我们还将继续开发更有效的协议和策略，用于序列数据的生成、收集、注释和质量控制，这将有助于工作草案和最终的小鼠序列。

The major goal of the work proposed is to continue expanding our Genome Center and to sequence at least 5 percent of the mouse genome to at least 99 percent accuracy for a working draft of approximately 5 fold coverage for random BACs from Dr. Pieter de Jong s mouse C57BL6/J BAC library. BACs of high biological interest supplied by the mouse community and verified by Dr. Reeves at Johns Hopkins University. This approximately 150 Mbp of mouse genomic sequence that includes approximately 30 Mbp at 99.99 percent accuracy that can be finished by a) primer walking off the pUC-based subclones and/or off the BAC, b) by multiplex PCR-based sequencing or c) a combination of both, and approximately 120 Mb or working draft sequence (greater than 99 percent accuracy) by the end of year 01. In year 02, an additional approximately 30 Mbp of finished and approximately 270 Mbp of working draft sequence will be generated. During year 03 we will concentrate on finishing the data generated in the first two years and/or generate additional working draft data in consultation with NHGRI staff and the NHGRI Advisory Committee. Approximately 10 percent of the clones will enter our sequencing queue as recommended by a Mouse Genome Oversite Committee and verified by Dr. Reeves at Johns Hopkins University supported via a sub-contract. Our center, which is built upon a successful parallel structured, graduate student/postdoctoral training-based approach to genomic sequencing, now has completed and deposited over 10 million bases of finished human and mouse genomic DNA sequence into GenBank at a total cost of less than 28 cents/base, calculated by total dollars in/total bases completed in GenBank. Our success has been based in part on a) our productive collaborations, with for example Dr. Reeves with whom we have completed almost 3 Mb of mouse genomic DNA sequence, b) our organizational schema which has resulted in improved training methods and more efficient sequencing strategies and methods, and c) our effective implementation of an inexpensive primer-directed MultiPlex PCR-based closure strategy. We plan an initial 50 percent expansion of our staff and a doubling of our sequencing related equipment and supplies in year 01, a similar increase in 02, and smaller increases in year 03. In anticipation of this expansion, our laboratory now has been re-organized into 4 intertwined groups, Management, Support, Research and Sequencing, with 15 parallel sequence production groups that are prepared convert from human or bacterial to mouse genomic sequencing. We also will continue developing more efficient protocols and strategies for sequence data generation, collection, annotation, and quality control that will contribute to both the working draft and final mouse sequence.