High throughput single molecule approaches for phased genome sequence assembly

项目总结/摘要 基于单分子的联合收割机技术有可能实现从头基因组 序列组装与定相和全基因组结构变异鉴定。从头 组装的全基因组测序完全描述了二倍体人类基因组， 少数长的、高度重复的序列，如着丝粒、端粒和 几乎相同的片段复制。基因组定相测序成功的关键 组装是我们小组最初开发的单分子作图方法， 现在由Bionano Genomics改进。所述方法从长链寡核苷酸的序列特异性标记开始， (180 kb至>1 Mb），具有荧光团的双链基因组DNA片段，随后是高- 线性化荧光DNA分子的自动成像和分析， 纳米通道阵列上的可商购的仪器。在下一阶段， 项目，我们建议生产2个个体的分阶段基因组序列组装， 1000个基因组计划的所有26个种族中的每一个都作为一般参考， 社区此外，我们还将进一步发展单分子标记技术， 难以在全基因组范围内进行询问并精确定相的重复元件- 范围目标区域。我们正在采取的方法来重新构建分阶段和组装 基因组将以高效率和低成本产生“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，并提供进一步的洞察力， 人类群体的结构变异模式。技术开发目标 这项建议将解决当今基因组分析所面临的一些最困难的问题。在 这个为期四年的项目结束时，一个强大的方法，分阶段基因组组装，重复 序列作图和远程定相将被开发，并准备在许多领域应用。 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.