Molecular Tools for Genome Partitioning

用于基因组分区的分子工具

• 批准号: 7509183
• 负责人: Jay Ashok Shendure
• 金额: $ 21.5万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-23 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7509183
• 关键词: Address; Algorithms; Area; Biological; Candidate Disease Gene; Clinical; Code; Compatible; Complement; Complex; DNA Resequencing; DNA Sequence; Development; Diagnostic; Disease; Drops; Exons; Experimental Designs; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Research; Genetic Variation; Genome; Genomics; Genotype; Goals; Gold; Human; Human Genome; Individual; Length; Libraries; Malignant Neoplasms; Maps; Medical; Medicine; Methods; Molecular; Numbers; Oligonucleotides; Pathway interactions; Performance; Personal Satisfaction; Polymerase Chain Reaction; Positioning Attribute; Proteins; Protocols documentation; Public Health; Range; Reaction; Reading; Research; Research Personnel; Role; Sample Size; Shotgun Sequencing; Somatic Mutation; Specificity; Standards of Weights and Measures; Technology; Variant; Work; base; concept; cost; design; disease phenotype; genetic linkage analysis; genome wide association study; human disease; improved; interest; mammalian genome; next generation; novel; novel strategies; research study; tool

DESCRIPTION (provided by applicant): A new generation of technologies is poised to reduce the cost of DNA sequencing by over two orders of magnitude. However, the routine sequencing of full human genomes will continue to be prohibitively expensive in the context of studies that require even modest sample sizes. However, it is frequently the case that investigators are interested in identifying germline variation or somatic mutations in a particular subset of the genome. Examples of genomic subsets that are highly relevant in the context of specific studies include: (a) a locus to which a disease phenotype has been mapped (i.e. a contiguous genomic region); and (b) the exons of genes belonging to a specific disease-related pathway (i.e. a large set of short, discontiguous sequences). Such subsets total to megabases in length, raising the question of how they can be efficiently isolated without performing hundreds to thousands of PCR reactions per genome. Our ability to take advantage of the power of next-generation sequencing technologies is markedly impaired by the lack of a corresponding targeting method, analogous to PCR that is matched to the scale at which the new sequencing platforms will routinely operate. To address this critical need, we will explore several novel strategies for "genome partitioning". Our goal is to develop these strategies into broadly available methods that enable the selective and uniform amplification of complex, arbitrary subsets of a mammalian genome in a single reaction. Our specific aims are: (1) to develop an enzymatic method for the uniform amplification of large sets of exon sequences from a human genome; (2) to develop a hybridization-based method for the selective amplification of contiguous megabase-scale regions from a human genome; (3) to integrate these methods with next-generation sequencing technologies, validating their utility by performing targeted variation discovery in a small number of individuals. PUBLIC HEALTH RELEVANCE: As we enter an era of "personalized medicine", DNA sequencing technology will be increasingly important to public health, contributing towards the unraveling of the genetic basis of human disease, as well as serving an increasing role in clinical diagnostics. Next-generation sequencing technologies have the potential to markedly accelerate genetics research, but are markedly hindered by the lack of equivalently powerful methods to target specific subsets of the human genome. We propose here to develop technologies that meet this critical need.

描述（由申请人提供）：新一代技术有望将DNA测序的成本降低两个数量级以上。然而，对人类全基因组的常规测序在需要甚至适度样本量的研究背景下仍将是极其昂贵的。然而，通常情况下，研究人员感兴趣的是鉴定基因组特定子集中的种系变异或体细胞突变。在特定研究的背景下高度相关的基因组子集的实例包括：（a）疾病表型已经映射到的基因座（即，连续的基因组区域）;和（B）属于特定疾病相关途径的基因的外显子（即，一大组短的不连续序列）。这些子集的长度总计为百万碱基，这就提出了一个问题，即如何在不对每个基因组进行数百至数千次PCR反应的情况下有效地分离它们。我们利用下一代测序技术的能力由于缺乏相应的靶向方法而明显受损，类似于与新测序平台常规操作规模相匹配的PCR。为了满足这一关键需求，我们将探索几种新的“基因组分区”策略。我们的目标是将这些策略发展成广泛可用的方法，使在单一反应中选择性和均匀扩增哺乳动物基因组的复杂的任意子集。我们的具体目标是：（1）开发用于从人类基因组均匀扩增大量外显子序列的酶促方法;（2）开发用于从人类基因组选择性扩增连续兆碱基规模区域的基于杂交的方法;（3）将这些方法与下一代测序技术整合，通过在少量个体中进行靶向变异发现来验证其效用。 公共卫生关系：随着我们进入“个性化医疗”时代，DNA测序技术对公共卫生将越来越重要，有助于揭示人类疾病的遗传基础，并在临床诊断中发挥越来越大的作用。下一代测序技术有可能显著加速遗传学研究，但由于缺乏针对人类基因组特定子集的同等强大方法而受到明显阻碍。我们在这里建议开发满足这一关键需求的技术。