OKLAHOMA UNIVERSITY GENOME CENTER - ADVANCED CENTER FOR

点击翻译按钮获取中文摘要

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.