Massively parallel contiguity mapping

大规模并行邻接映射

• 批准号: 8181132
• 负责人: Jay Ashok Shendure
• 金额: $ 58.59万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8181132
• 关键词: Address; Cells; Clinical; Complex; DNA; DNA Resequencing; DNA Sequence; Development; Diagnostic; Emulsions; Event; Generations; Genetic; Genome; Genomics; Goals; Haplotypes; Human Genome; In Situ; In Vitro; Individual; Length; Libraries; Ligation; Maps; Medicine; Methods; Molecular; Optics; Pattern; Principal Investigator; Production; Public Health; Reading; Reagent; Recovery; Relative (related person); Research; Role; Shotgun Sequencing; Shotguns; Source; Stretching; Surface; Technology; Transposase; Work; base; blind; cost; cost effective; density; human disease; innovation; mammalian genome; method development; mouse genome; novel; prevent; restriction enzyme; scaffold; technology development

DESCRIPTION (provided by applicant): Massively parallel technologies have reduced the per-base cost of DNA sequencing by several orders of magnitude. However, limited read lengths and a lack of methods to establish contiguity over even modest distances have prevented these technologies from achieving the high-quality, low-cost de novo assembly of mammalian genomes. Even as revolutionary sequencing technologies further mature, it may continue to be the case that the best technologies in terms of cost-per-base yield reads that are of an insufficient length or quality for the effective de novo assembly of large genomes. To address this critical need, we are exploiting high density, random, in vitro transposition as a novel means of physically shattering genomic DNA in creative ways that facilitate the recovery of contiguity information at different scales. Our project is divided into four aims, the first three of which are respectively directed at the development of massively parallel methods for determining short-range, mid-range, and long-range contiguity. These are: 1) a method for shattering genomic DNA with symmetric tags that post hoc inform the ordering of adjacent fragmentation events in a way that is entirely independent of the primary sequence content; 2) a method for massively parallel, in vitro barcoding of fosmid or BAC-sized subsequences of a genome, thereby facilitating hierarchical assembly; 3) an in situ method for converting stretched DNA molecules into adaptor-flanked libraries, such that reads generated by massively parallel sequencing will remain linearly ordered in terms of the XY coordinates at which they originate. In the fourth aim, we will integrate these methods to demonstrate: 1) the highly cost-effective de novo assembly of the mouse genome with a quality that exceeds that of the original assembly; 2) the highly cost-effective haplotype resolved resequencing of a human genome. PUBLIC HEALTH RELEVANCE: As we enter an era of personalized medicine, a deep understanding of the human genome will be increasingly important to public health, contributing to the unraveling of the genetic basis of human disease, as well as serving an increasing role in clinical diagnostics. The technologies developed by this project will accelerate progress towards these goals by enabling the affordable sequencing of haplotype-resolved human genomes. These same technologies will also facilitate the high-quality, cost-effective assembly of the genomes of other mammalian species, which inform our understanding of the human genome through evolutionary analysis.

描述（由申请人提供）：大规模并行技术已经将DNA测序的每碱基成本降低了几个数量级。然而，有限的读取长度和缺乏在甚至适度的距离上建立邻接的方法已经阻止了这些技术实现哺乳动物基因组的高质量、低成本的从头组装。即使革命性的测序技术进一步成熟，就每碱基成本而言，最好的技术产生的读段长度或质量对于大基因组的有效从头组装来说可能仍然不足。为了满足这一关键需求，我们正在利用高密度，随机，体外转座作为一种新的手段，以创造性的方式物理粉碎基因组DNA，促进在不同尺度上恢复邻接信息。我们的项目分为四个目标，其中前三个分别针对大规模并行方法的发展，以确定短距离，中距离和长距离的邻接。这些是：1）用于粉碎具有对称标签的基因组DNA的方法，所述对称标签以完全独立于一级序列含量的方式事后通知相邻片段化事件的排序; 3）用于将拉伸的DNA分子转化为衔接子侧翼文库的原位方法，使得通过大规模平行测序产生的读段将在它们起源的XY坐标方面保持线性有序。在第四个目标中，我们将整合这些方法来证明：1）小鼠基因组的高成本效益从头组装，其质量超过原始组装; 2）人类基因组的高成本效益单倍型解析重测序。 公共卫生关系：随着我们进入个性化医疗时代，对人类基因组的深入了解将对公共卫生越来越重要，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。该项目开发的技术将加快实现这些目标的进展，使人们能够负担得起的单体型分辨人类基因组测序。这些技术还将促进其他哺乳动物物种基因组的高质量、高成本效益的组装，这将通过进化分析为我们了解人类基因组提供信息。