A Strategy for High Quality Clinical Resequencing of the Human Genome

描述（由申请人提供）：本提案的总体目标是制定人类基因组临床重测序的策略，我们将其定义为将人类基因组重测序应用于大型临床人群，用于疾病基因发现研究以及具有诊断潜力的临床应用。我们的策略依赖于太平洋生物科学平台所使用的“第三代”单分子DNA测序。我们预计，单分子DNA测序技术最终将成为测序仪的选择，因为这些系统可以快速收集大量患者样本的DNA序列，并附有临床结果注释，并且成本低廉，操作成本仅为其他测序技术的一小部分。第一个目标侧重于开发我们称之为间歇性片段重测序的方法。从单个DNA分子中产生一组分散的reads，与配对序列的工作方式非常相似，但具有内部间隔序列，而不是由成对末端方法产生的末端序列对。第二个目标涉及测试基于溶液的方法，以结合基因组霰弹枪测序，大幅增加具有高度临床意义的基因组学区域的覆盖率。鉴于序列的随机分布，霰弹枪基因组测序工作对基因组的所有区域一视同仁。与基因组的其余部分相比，选择性地增加这些感兴趣区域的折叠覆盖率，将提高对重要临床变异的检测。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop a strategy towards clinical resequencing of the human genome which we define as the application of human genome resequencing to large clinical populations for disease gene discovery research as well as a clinical application that has diagnostic potential. Our strategy relies on "third generation" single molecule DNA sequencing as utilized by the Pacific Biosciences platform. We anticipate that single molecule DNA sequencing technology will ultimately be the sequencer of choice given these systems' rapid collection of massive amounts of DNA sequences from patient samples with clinical outcome annotation, and cost-effective operation at a fraction of the current costs of other sequencing technologies. This first goal focuses on developing what we term an intermittent segment resequencing approach. A set of interspersed reads will be generated from a single DNA molecule, much the same way as mate paired sequence works, but having internal interval sequences rather than a terminal sequence pair as generated by paired-end approaches. The second goal involves testing in-solution based approaches to increase substantially the fold coverage of genomics regions of high clinical interest in combination with genome shotgun sequencing. Given their random distribution of sequences, shotgun genome sequencing efforts treat all regions of the genome equally. Increasing the fold-coverage of these regions of interest selectively, in comparison to the rest of the genome, would improve the detection of important clinical variants. PUBLIC HEALTH RELEVANCE: Creating a "personalized" medicine strategy for each individual requires decoding the DNA sequence of a person's cancer that involves identifying genetic variations for any patient and discovering the clinical significance of these variants. The composition and spectrum of these variants can vary significantly from individual to individual, thus pointing out the importance of identifying all of the critical ones with clinical ramifications. New sequencing technologies have opened the way for creating personalized genetic signatures which can be used to tailor the clinical management of individuals but even more cost-effective approaches are needed given that thousands of individuals with specific disease have their genomes sequenced.