Molecular Tools for Genome Partitioning

描述(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，在需要适度样本量的研究背景下，对全人类基因组的常规测序将继续昂贵得令人望而却步。然而，通常的情况是，研究人员对识别基因组特定子集的种系变异或体细胞突变感兴趣。在具体研究背景下高度相关的基因组亚集的例子包括：(A)疾病表型被映射到的一个基因座(即一个连续的基因组区域)；以及(B)属于特定疾病相关途径的基因的外显子(即一大组短的、不连续的序列)。这些亚群的总长度达到百万碱基，这引发了一个问题，即如何在不对每个基因组进行数百到数千次PCR反应的情况下有效地分离它们。由于缺乏相应的靶向方法，我们利用下一代测序技术的能力明显受损，这类似于与新测序平台将常规操作的规模相匹配的聚合酶链式反应。为了解决这一关键需求，我们将探索几种新的“基因组分割”策略。我们的目标是将这些策略发展成广泛可用的方法，使之能够在单一反应中选择性和统一地扩增哺乳动物基因组的复杂的、任意的子集。我们的具体目标是：(1)开发一种从人类基因组中统一扩增大量外显子序列的酶方法；(2)开发一种基于杂交的方法，从人类基因组中选择性扩增连续的巨型碱基区域；(3)将这些方法与下一代测序技术相结合，通过在少数个体中进行有针对性的变异发现来验证它们的有效性。 与公共健康相关：随着我们进入“个性化医学”时代，DNA测序技术对公共健康将变得越来越重要，有助于解开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。下一代测序技术具有显著加快遗传学研究的潜力，但由于缺乏同等强大的方法来针对人类基因组的特定亚组，因此明显受到阻碍。我们在这里建议开发满足这一关键需求的技术。

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.