HIGH THROUGHPUT MULTIPLEX SEQUENCING

这个项目的目标是定义计算机辅助的能力 多重测序技术在双胞胎基因组中的全面应用 生物医学上的重要生物：麻风分枝杆菌和麻风杆菌。 肺结核。这些生物代表着一个主要的世界健康问题， 导致的死亡人数超过任何其他感染性病原体。它们的基因组DNA 序列将提供一个无价的资源，Rational将在 疾病控制可以建立在。此外，与成熟的 基因能力，这些序列将提供一个独特的机会 探索这些复杂病原体的基本生物学。每个基因组都将是 在粘粒的基础上进行排序，从一组有序的 克隆人。麻风杆菌基因组将首先完成，因为一组绘制的 宇宙星已经存在。三年后麻风杆菌的序列将是 已经完成，结核分枝杆菌的工作将开始。通过技术 改进，强调质量控制，并始终如一 生产努力，测序能力超过4Mb的粘粒 每年的序列将在该项目的最后两年开发。 在整个过程中，每个基地的总成本将低于50美分。 数据将通过数字胶片扫描来收集。序列将被读取并 使用我们开发的复制品软件包组装成重叠群 合作者，G·丘奇博士及其在哈佛医学院的同事。这个 结果将显示在改装的GelAssembly平台上， 交互式编辑，可即时访问存储的胶片图像。 当两条链都已经完成时，序列将被认为是“完成的” 完全测序，错误率不超过1/1000个核苷酸。 这一水平的准确性将通过随机散弹枪排序来实现 平均深度为7倍冗余。在这种程度的覆盖下，很少或 没有完成工作将是必要的。然而，我们正在开发一种聚合酶链式反应- 基于的方法来简化可能需要的任何完成测序 搞定了。我们产生的所有数据都将被输入到分枝杆菌 序列数据库和Genbank。对基因序列的分析， 有趣的特征、调控元素、进化保守等。 将由分枝杆菌研究社区完成，并得到 序列数据库和与之密切相关的合作者网络 这个项目。

The goal of this project is to define the capabilities of computer-assisted multiplex sequencing by full scale application to the genomes of two biomedically important organisms: Mycobacterium leprae and M. tuberculosis. These organisms represent a major world health problem, causing more deaths than any other infectious agent. Their genomic DNA sequences will provide an invaluable resource on which rational efforts at disease control can be based. Moreover, combined with sophisticated genetic capabilities, the sequences will provide a unique opportunity to explore the basic biology of these complex pathogens. Each genome will be sequenced on a cosmid by cosmid basis starting with an ordered set of clones. The M. leprae genome will be done first since a mapped set of cosmids already exists. In three years the M. leprae sequence will be finished and work on M. tuberculosis will begin. Through technical improvements, a strong emphasis on quality control, and consistent production efforts, sequencing capabilities exceeding 4 Mb of cosmid sequences per year will be developed in the last 2 years of the project. This throughout will be achieved at a total cost under 50 cents per base. Data will be gathered by digital film scanning. Sequences will be read and assembled into contigs using the REPLICA software package developed by our collaborator, Dr. G. Church and associates at Harvard Medical School. The results will be displayed on a modified GelAssemble platform for rapid, interactive editing with instantaneous access to stored film images. Sequences will be considered "finished" when both strands have been completely sequenced at an error rate not exceeding 1 in 1000 nucleotides. This level of accuracy will be achieved by random shotgun sequencing to a mean depth of 7-fold redundancy. With this degree of coverage, little or no finishing work will be necessary. However, we are developing a PCR- based method to facilitate any finish sequencing that might need to be done. All of the data we produce will be entered into a mycobacterial sequence database as well as Genbank. Analysis of the sequences for genes, interesting features, regulatory elements, evolutionary conservation, etc. will be done by the mycobacteria research community, assisted by the sequence database and a network of collaborators closely associated with the project.