SEQUENCING OF HUMAN CHROMOSOME 16P

人类身体测绘工作和自动化DNA的现状 测序已经使考虑开始对 人类基因组正以相当快的速度增长。YAC/COSMID综合物理 在洛斯阿拉莫斯国家实验室构建的16号染色体图谱是 在最完整的人类染色体图谱中。的40 MBP p臂 16号染色体提供了一个很好的起点来测试 将100kb分辨率的物理地图转换为集合的有效性 用于测序的最小重叠克隆，并最终用于 获得染色体的完整序列。近乎完整的YAC 16号染色体的MAP是可用的，因为它是一组广泛的宇宙和 STSS定位于YAC图谱的粘粒重叠群平均间距为150kb 分开。这里提出的战略将基于确定BAC、PAC、 和其他稳定的、大插入的克隆，从16p中选择非 用于初始测序的重叠集，并开发最小 克隆的平铺路径以使测序达到闭合。在整个过程中 项目，将进行技术开发，强调增加 高质量序列数据的生成速度和降低成本。 第一步将是确定一组最小重叠或不重叠的 覆盖人类染色体16p的重叠BAC克隆 先前建立的染色体16p图谱。我们估计大约有 200个这样的BAC将覆盖16个短臂中的至少70%。 克隆将立即用于测序。这将是必要的 BAC鸟枪式测序文库制作方法的研究进展 克隆，以便可以快速有效地进行测序。非- 从现有的大重叠群中重叠的BAC和粘粒克隆 16号染色体图谱将使用随机散弹枪策略进行测序，该策略 生产高质量的成品序列。16便士的绝大多数可以是 用这种方法测序。使用YAC关闭地图的策略， BAC、PAC、P1和粘粒克隆和完整序列的开发- 还将开发和测试16P的现成克隆集。几个 与自动测序相关的软件增强功能将包括 在样本跟踪和数据管理方面有所改进， 序列组装和将碱基调用置信值纳入 组装和编辑。技术发展的结果和 软件改进将把测序成本从0.30美元降低到 每对已完成的碱基对在三年内降至0.18美元。准确，带注释 数据将在六个月内提供给科学界 完成每一段。

The current status of human physical mapping efforts and automated DNA sequencing have made it feasible to consider beginning to sequence the human genome at a substantial pace. The integrated YAC/cosmid physical map constructed at Los Alamos National Laboratory for chromosome 16 is among the most complete of the human chromosome maps. The 40 Mbp p arm of chromosome 16 presents an excellent starting point to test the effectiveness of converting a physical map at 100 kb resolution into a set of minimally overlapping clones for sequencing and ultimately for obtaining the complete sequence of the chromosome. A nearly complete YAC map is available for chromosome 16 as is an extensive set of cosmids and cosmid contigs anchored to the YAC map by STSs spaced an average of 150 kb apart. The strategy proposed here will be based on identifying BAC, PAC, and other stable, large-insert clones from 16p, selecting a non- overlapping set for initial sequencing, and development of a minimal tiling path of clones for sequencing to reach closure. Throughout the project, technology development will be performed to stress increasing the rate of generation of high quality sequence data and decreasing the cost. The first step will be to identify a set of minimally overlapping or non- overlapping BAC clones that cover human chromosome 16p using the previously established chromosome 16p map. We estimate that approximately 200 such BACs will cover at least 70% of the short arm of 16. These clones will be used immediately for sequencing. This will necessitate development of methods for making shotgun sequencing libraries from BAC clones so that they may be rapidly and efficiently sequenced. Non- overlapping BACs and cosmid clones from large contigs in the existing chromosome 16 map will be sequenced using a random shotgun strategy which produces high quality finished sequence. The great majority of 16p can be sequenced by this approach. Strategies for closure of the map using YAC, BAC, PAC, P1, and cosmid clones and development of a complete sequence- ready clone set for 16p will also be developed and tested. Several enhancements in the software associated with automated sequencing will be made including improvements in sample tracking and data management, sequence assembly, and incorporation of base-calling confidence values in assembly and editing. The result of the technology development and software improvements will be to reduce the cost of sequencing from $0.30 to $0.18 per finished base pair over three years. Accurate, annotated data will be provided to the scientific community within six months of completion of each segment.