Bacterial Genome Diversity

该项目的总体目标是表征与不同分离株相关的基因组序列， 鼠疫耶尔森氏菌、类鼻疽伯克霍尔德氏菌和土拉热弗朗西丝氏菌在综合水平上， 但对任何细菌物种都是如此。常规的全基因组测序将在 每个物种至少增加一个菌株，以补充现有项目的数据。但主要 重点将放在对大量菌株的基因组片段进行靶向重测序上。拟议 这项技术依赖于fosmid克隆，一种适合克隆基因组片段的重组DNA系统 大小约为40 kbp。随机挑选的磷酰胺将通过末端测序表征， 限制性消化指纹图谱;将分析足够数量的克隆以提供深度（约100%）的DNA序列。 10 X）分析的每种细菌菌株的全基因组覆盖率。Fosmid末端序列的比对和 具有可用参考序列的引物将允许检测新菌株中的基因组区域， 相对于参考序列高度发散或重排。来自这些地区的化石将 完全排序。还将对数据进行分析，以全面了解 分析每个菌株的整个基因组的遗传变异。 我们希望该项目能够为深入分析主要细菌的基因组提供新的标准。 物种你！最后，目标将是建立一个足够大和多样化的参考资料数据库， 序列--一些是整个基因组，另一些是高度可变的基因组片段--大多数基因和基因 在新分离的菌株中遇到的排列将在数据库中表示。

The overall goal of this project is to characterize the genome sequences associated with diverse isolates of Yersinia pestis, Burkholderia pseudomallei, and Francisella tularensis at a level of comprehensiveness not yet achieved for any bacterial species. Conventional whole-genome sequencing will be carried out on at least one additional strain of each species to complement data from existing projects. However, the main focus will be on targeted resequencing of genome segments from large numbers of strains. The proposed technology relies on fosmid clones, a recombinant DNA system suitable for cloning genome segments approximately 40 kbp in size. Randomly picked fosmids will be characterized by end-sequencing and restriction-digest fingerprinting; a sufficient number of clones will be analyzed to provide deep (approx. 10X) whole-genome coverage of each bacterial strain analyzed. Alignment of fosmid end sequences and digests with available reference sequences will allow detection of genomic regions in new strains that are highly diverged or rearranged relative to the reference sequences. Fosmids from these regions will be completely sequenced. The data will also be analyzed to obtain a comprehensive view of the patterns of genetic variation across the whole genome of each strain analyzed. We expect the project to exemplify a new standard for thorough analysis of the genomes of major bacterial species. U!timately, the goal will be to create a sufficiently large and diverse database of reference sequences--some of whole genomes others of highly variable genome segments--that most genes and gene arrangements encountered in newly isolated strains will be represented in the database.