Northwest Genomics Center

项目摘要：本申请涉及NHLBI- rfa - od -09-004，用于对表型良好的NHLBI队列进行大规模DNA测序和分子分析。我们建议建立一个测序中心，对来自表型良好的NHLBI队列的10,000个基因组DNA样本进行生产水平的外显子组重测序。第二代靶向捕获和DNA测序方法已经迅速成熟。外显子组测序目前比全基因组测序在研究罕见变异对心脏、肺和血液疾病的贡献方面具有优势。这些优点包括每个样本的成本低得多，并且更有可能识别出适合功能解释的大影响变量。在我们的初步研究中，我们开发了在全基因组范围内（即外显子组）靶向捕获和第二代蛋白质编码序列测序的方法。我们始终能够在96%的目标碱基上识别编码变异，而整个基因组测序所需的工作量仅为5%。结果是高质量的外显子组，与基因型的一致性为99.75%，新变异的错误发现率<1%。我们还展示了外显子组测序直接鉴定单基因疾病的致病基因的能力。这一概念验证可作为扩展外显子组测序以研究与NHLBI任务相关的极端和/或复杂表型的起点。在保持高数据质量的同时，提高吞吐量或降低成本的改进将集成到外显子组生产管道中。我们最近的创新包括一种新的算法，该算法可以从第二代测序图像集中提取的可用序列数据量几乎翻倍。我们团队的生产重点将辅以高通量测序和基因分型、技术开发（实验和算法）、罕见变异统计分析、群体遗传学和拷贝数变异方面的专家。样本将从NHLBI队列接收，并在外显子组测序之前进行广泛的质量控制。在测序之后，我们将为每个个体提供一组完整注释的编码变体。对于最终交付的产品，我们将开发一个定制的基因分型芯片，用于多达50,000个高影响，非同义变体，以在更大的队列样本集（多达50,000个）上进行分析。我们期待与队列研究者和NHLBI密切合作，以最大限度地发挥这些数据和这个项目的科学价值。

DESCRIPTION (provided by applicant): Project Abstract: This application addresses the NHLBI-RFA-OD-09-004 for Large-scale DNA Sequencing and Molecular Profiling of Well-phenotyped NHLBI Cohorts. We propose to establish a sequencing center to perform the production-level resequencing of exomes from 10,000 genomic DNA samples derived from well-phenotyped NHLBI cohorts. Second generation methods for targeted capture and DNA sequencing have matured rapidly. Exome sequencing currently has advantages over whole genome sequencing for studies aimed at understanding the contribution of rare variants to heart, lung and blood diseases. These advantages include much lower costs per sample and an increased likelihood of identifying variants of large effect that are amenable to functional interpretation. In our preliminary studies, we developed methods for targeted capture and second generation sequencing of protein-coding sequences at a genome-wide scale, i.e. the exome. We are consistently able to identify coding variants at 96% of targeted bases for 5% of the sequencing effort required for a whole genome. The result is high quality exomes, with a concordance to genotype calls of >99.75% and a false discovery rate for novel variants of <1%. We also show the power of exome sequencing for the direct identification of the causative gene for a monogenic disease. This proof-of-concept serves as a starting point for extending exome sequencing to study extreme and/or complex phenotypes of relevance to the NHLBI mission. Improvements that increase throughput or decrease costs while maintaining high data quality will be integrated into the exome production pipeline. Our recent innovations include a novel algorithm that nearly doubles the usable amount of sequence data that can be extracted from second generation sequencing image sets. The production focus of our team will be complemented by experts in high-throughput sequencing and genotyping, technology development (experimental and algorithmic), the statistical analysis of rare variation, population genetics, and copy number variation. Samples will be received from NHLBI cohorts and undergo extensive quality control prior to exome sequencing. Following sequencing, we will deliver a fully annotated set of coding variants for each individual. For the final deliverable, we will develop a custom genotyping chip for up to 50,000 high-impact, nonsynonymous variants to be assayed on a larger set of cohort samples (up to 50,000). We anticipate working closely with cohort investigators and the NHLBI to maximize the scientific value of these data and of this program. PUBLIC HEALTH RELEVANCE: Project Narrative: Well-phenotyped cohorts provide a key resource for studying the contribution of genetic variation to traits related to heart, lung or blood diseases. Applying targeted capture and massively parallel sequencing of all protein coding regions in the human genome (the exome) to well-phenotyped cohorts will help to delineate the contributions of both rare and common protein-altering variants to common diseases for the first time.