High throughput single molecule approaches for phased genome sequence assembly

项目摘要/摘要 基于单分子的组合技术获得从头开始的基因组是可能的 序列组装与分期和全基因组结构变异鉴定。从头开始 组装的全基因组测序完全描述了二倍体人类基因组，除了 少量的长的、高度重复的序列，如着丝粒、端粒和 近乎相同的节段性复制。阶段性基因组测序成功的关键 组装是我们团队最初开发的单分子图谱方法， 现在由Bionano基因组公司改进。该方法从Long的序列特定标记开始 (180kb到&gt；1Mb)，带有荧光团的双链基因组DNA片段，然后是高- 线性化荧光DNA分子的吞吐量、自动成像和分析 商业仪器上的纳米通道阵列。在此的下一阶段 项目中，我们建议从两个个体中产生阶段性基因组序列组合 1000基因组计划中所有26个民族中的每一个都作为 社区。此外，我们还将进一步发展单分子标记技术来绘制图谱 这些重复的元素很难在全基因组范围内进行讯问，也很难精确地进行长时间的相变- 射程目标区域。我们正在采取的方法是分阶段和组装地建造新的 基因组将以高效率和低成本生产出接近参考级的基因组 世界各地的许多民族。这些参考序列将大幅增加 已经获得的所有全基因组序列的价值并提供了进一步的洞察 人类群体的结构性变异模式。公司的技术发展目标 这项提案将解决当今基因组分析面临的一些最困难的问题。在… 这个为期四年的项目的结束，是一种稳健的阶段性基因组组装方法，重复性 序列映射和远程相移将被开发出来，并准备在许多方面应用 基因组研究领域。

Project Summary/Abstract It is possible to combine technologies based on single molecules to achieve de novo genome sequence assembly with phasing and genome-wide structural variation identification. De novo assembled whole genome sequencing fully describes the diploid human genome except for a small number of long, highly repetitive sequences such as the centromeres, telomeres, and near-identical segmental duplications. Key to the success of phased genome sequence assembly is the single molecule mapping approach originally developed by our group and is now improved by Bionano Genomics. The method starts with sequence-specific labeling of long (180 kb to >1 Mb), double-stranded genomic DNA fragments with fluorophores followed by high- throughput, automated imaging and analysis of the linearized fluorescent DNA molecules in nanochannel arrays on a commercially available instrument. During the next phase of this project, we propose to produce phased genome sequence assemblies of 2 individuals from each of all 26 ethnic groups of the 1000 Genomes Project to serve as general references for the community. In addition, we will further develop the single molecule labeling technology to map repetitive elements that are difficult to interrogate genome-wide and to precisely phase long- range target regions. The approach we are taking to construct de novo phased and assembled genomes will produce “near reference grade” genomes with high efficiency and at low cost for many ethnic groups around the world. These reference sequences will increase substantially the value of all the whole genome sequences already obtained and provide further insight into structural variation patterns across human populations. The technology development aims of this proposal will address some of the most difficult questions facing genome analysis today. At the end of this four-year project, a robust method for phased genome assembly, repetitive sequence mapping, and long-range phasing will be developed and ready for application in many areas of genome research.