SEQUENCING OF HUMAN CHROMOSOME 16P

人类物理绘图工作和自动化DNA的现状 测序使得考虑开始测序成为可能， 人类基因组的研究。 集成的YAC/粘粒物理 洛斯阿拉莫斯国家实验室构建的16号染色体图谱， 最完整的人类染色体图谱之一 40 Mbp的p臂 16号染色体提供了一个很好的起点来测试 将100 kb分辨率的实物地图转换为一组地图的有效性 用于测序的最小重叠克隆， 获得染色体的完整序列。 几乎完整的YAC 16号染色体有一张图，这是一套广泛的染色体组， 粘粒重叠群通过平均间隔150 kb的STS锚定到YAC图谱上 apart. 这里提出的战略将基于确定BAC，PAC， 和其他稳定的，大插入克隆从16 p，选择一个非- 重叠集的初始测序，并制定一个最小的 平铺克隆路径以进行测序以达到闭合。 在整个 项目，将进行技术开发，以强调增加 提高了高质量序列数据的生成速率，降低了成本。 第一步将是确定一组最低限度重叠或不重叠的 重叠BAC克隆，覆盖人类染色体16 p，使用 先前建立的染色体16 p图谱。 我们估计， 200个这样的BAC将覆盖16个短臂的至少70%。 这些 克隆将立即用于测序。 这将需要 从BAC制备鸟枪测序文库的方法的开发 克隆，以便它们可以快速有效地测序。 非 来自现有的大重叠群的重叠BAC和粘粒克隆， 16号染色体图谱将使用随机鸟枪策略进行测序， 生产高质量的成品序列。 16 p的绝大多数可以 通过这种方式排序。 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16 p的现成克隆集。 几 与自动测序相关的软件增强将 包括改进样品跟踪和数据管理， 序列组装，并将碱基识别置信度值并入 组装和编辑。 技术发展的结果， 软件改进将把测序成本从0.30美元 每完成一个碱基对的价格是0.18美元。 准确，有注释 数据将在六个月内提供给科学界。 完成每一个环节。

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.