OKLAHOMA UNIVERSITY GENOME CENTER - ADVANCED CENTER FOR

拟议工作的主要目标是继续扩大我们的基因组中心，并对来自Pieter de Jong S博士的C57BL6/J BAC文库的随机BAC进行至少5%的小鼠基因组测序，以至少99%的准确率进行随机BAC的工作草案。具有很高生物学价值的细菌由老鼠社区提供，并得到约翰·霍普金斯大学的里夫斯博士的验证。这个大约150 Mbp的小鼠基因组序列包括大约30Mbp的99.99%准确率的序列，可以通过a)走出基于PUC的亚克隆和/或离开BAC的引物，b)通过基于多重PCR的测序或c)两者的组合，以及到2001年底大约120 Mb或工作草稿序列(大于99%的准确率)来完成。在2002年，将产生另外约30Mbp的已完成序列和约270Mbp的工作草案序列。在2003年，我们将集中精力完成头两年生成的数据和/或与NHGRI工作人员和NHGRI咨询委员会协商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，这是由一个小鼠基因组超现场委员会推荐的，并由约翰·霍普金斯大学的里夫斯博士通过分包合同进行验证。我们的中心建立在成功的并行结构、研究生/博士后培训为基础的基因组测序方法的基础上，现在已经完成了1000多万个已完成的人类和小鼠基因组DNA序列，并将其存入GenBank，总成本不到28美分/个碱基，以总美元/在GenBank中完成的总碱基计算。我们的成功在一定程度上得益于a)我们与里夫斯博士的卓有成效的合作，例如，我们与他一起完成了近3Mb的小鼠基因组DNA序列；b)我们的组织架构带来了改进的培训方法和更高效的测序策略和方法；以及c)我们有效地实施了廉价的引导型多重PCR闭合策略。我们计划在01年将我们的员工人数增加50%，并将与测序相关的设备和用品增加一倍，在02年增加类似的数量，在03年增加较小的数量。在预料到这一扩大的情况下，我们的实验室现已重组为管理、支持、研究和测序4个相互交织的小组，并准备了15个平行序列产生小组，这些小组从人或细菌到小鼠基因组测序。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鼠标序列。

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.