CONTINUING MEGABASE SEQUENCING ON THE HUMAN X CHROMOSOME

来自人类X染色体的30兆碱基的DNA序列将被 在三年内产生的成本低于35美分， 基地这些数据将是高质量的，每一个错误都少于一个。 一万个核苷酸来自Xq28和Xp22的高分辨率粘粒图 将通过自动限制性内切酶消化产生区域 映射.将通过平衡随机和 基于亚克隆的双末端测序的定向策略， 将最小化冗余和对昂贵寡核苷酸的依赖 散步，避免过多的重复序列中的重叠 地区在粘粒地图的差距和模糊性将解决， 分离替代基因组克隆，并通过协调正在进行的部分 排序和映射。新技术提高大规模 将制定排序。高质量的简化施工 鸟枪文库和快速DNA模板制备方案将允许 前端步骤的部分自动化。新型荧光染料标记 寡核苷酸引物将能够实现流线型的序列反应， 产生的信号增强将简化DNA碱基识别。的 信息学基础设施，以协调 粘粒作图、DNA测序、数据组装、验证、注释和 数据库的提交将得到简化，以提高处理量。在 除了DNA序列，这个项目将产生一个模型， 一个扩展的程序，可以让整个人类及时测序， 基因组

Thirty megabases of DNA sequence from the human X chromosome will be generated over three years at a cost of less than 35 cents per finished base. The data will be of high quality, with less than one error per 10,000 nucleotides. High resolution cosmid maps from the Xq28 and Xp22 regions will be generated by automated restriction endonuclease digestion mapping. Selected cosmids will be analyzed by a balanced random and directed strategy based upon double ended sequencing of subclones that will minimize redundancy and dependence on expensive oligonucleotide walks, and avoid excessive redundant sequencing in the overlapping regions. Gaps and ambiguities in the cosmid maps will be solved by isolating alternative genomic clones, and by coordinating ongoing partial sequencing with the mapping. New technologies to improve large scale sequencing will be developed. Simplified construction of high quality shotgun libraries and a rapid DNA template preparation scheme will allow partial automation of front end steps. Novel fluorescent dye labeled oligonucleotide primers will enable streamlined sequence reactions and the resulting signal enhancements will simplify DNA base calling. The informatics infrastructure for the coordination of all steps from the cosmid mapping, DNA sequencing, data assembly, validation, annotation and database submission will be streamlined to allow higher throughput. In addition to the DNA sequence, this project will generate a model for an expanded program that can allow timely sequencing of the whole human genome.