Sequencing and Clone Characterization

项目2(测序和克隆表征)将产生总体 计划项目。它的基本任务是获取在项目1中确定的可能包含 人类基因组的新结构变体，并通过分层程序对其进行表征。 这一过程中的步骤将包括使用多种限制性内切酶进行指纹识别，光枪 测序，全鸟枪测序，最后完成。在每一步，化石都有可能 正确地标记为包含相对于人类参考序列的结构变体，将是 已评估。 项目2的第二项活动将是鉴定具有两个高质量末端序列的克隆， 这两个序列都与人类参考序列不一致。这个班的福斯米德要么是候选人 落在参考序列中的空隙中或相对于它的大插入中。项目2将构建重叠群 并选择跨越重叠群的最小平铺路径进行测序，从而提供 这一能力是可用的；或者，我们将带头寻找其他选项来排序 这些小路。在项目2中将优先考虑插入到参考序列中的克隆， 而不是从差距中，与计划项目的总体目标保持一致。 对人类基因组中等规模变异序列的完整定义将直接 在人类遗传学中的应用。与更深入研究的单核苷酸多态不同 (SNPs)，这些变异通常会影响数千或更多核苷酸的排列；因此，任何 给定的中等规模的变异比SNP更有可能改变一个或多个人类基因的功能。 变种的完整序列将在项目2中确定，这将允许可靠的设计 项目3中的基因分型分析可用于评估大范围内变异的存在或不存在 例如，在一系列病例和对照中确定的个体数量，以研究遗传 对某一特定疾病易感性的影响。

Project 2 (Sequencing and Clone Characterization) will generate much of the data produced by the overall Program Project. Its basic mission is to take fosmid clones, identified in Project 1 as being likely to contain novel structural variants of the human genome, and characterizethem through a hierarchical procedure. The steps in this procedure will include fingerprinting with multiple restriction enzymes, light-shotgun sequencing, full-shotgun sequencing, and finishing. At each step, the likelihood that the fosmids have been correctly flagged as containing structural variants relative to the human-reference sequence, will be evaluated. A second activity in Project 2 will be to characterize clones that have two good-quality end sequences, neither of which aligns with the human-reference sequence. Fosmids in this class are candidates either for falling in gaps in the reference sequence or within large insertions relative to it. Project 2 will build contigs from these "no hitter" clones and choose a minimal tiling path across the contigs for sequencing, providing that capacity is available; alternately, we will take the lead in finding other options for the sequencing of these paths. Priority within Project 2 will be given to clones from insertions into the reference sequence, rather than from gaps, in keeping with the overall goals of the Program Project. Complete definition of the sequences of intermediate-scale variants of the human genome will have direct applications in human genetics. Unlike the more intensively studied single-nucleotide polymorphisms (SNPs), these variants will typically affect the arrangement of thousands or more nucleotides; hence, any given intermediate-scale variant is more likely than a SNP to alter the function of one or more human genes. The complete sequences of the variants, which will be determined in Project 2, will allow design of reliable genotyping assays in Project 3 that can be used to assess the presence or absence of the variant in large numbers of individualsfor example, in a series of cases and controls ascertained to study genetic influences on susceptibility to a particular disease.