New Tools for Structural Variation Analysis, De Novo Assembly and Closing of Complex Genomes

描述(由申请人提供)：下一代测序(NGS)平台通过以低成本、高通量的格式提供海量数据，正在从根本上改变遗传和基因组研究。现有技术的主要缺点是它们产生的短序列读取长度。现有的文库制备方法在产生仅几kb的短插入片段方面也受到限制。因此，仅用短读NGS技术对基因组进行从头组装是不现实的。即使有一个高质量的参考人类基因组，在分析复杂的基因组区域时，重新测序和组装新的人类基因组也是一个巨大的挑战。如果不借助克隆的DNA，就无法实现超过几kb的单倍体分析。显然，需要新的工具来弥合大规模并行短读测序技术(1,500个碱基)和组装基因组所需的大支架&20 kb之间的差距。SBIR第一阶段赠款提案“复杂基因组的结构变异分析、新组装和关闭的新工具”建议开发一个新的NGS“前端”和支持它的软件。从大的随机剪切的DNA片段中构建无克隆的20-40kb的配对文库的技术还不存在。这项技术将使复杂基因组的准确组装成为可能，就像传统的基于克隆的策略中的Fosid和BAC末端序列一样。这些工具的开发可以减少基因组组装的人工关闭成本和计算成本，从而产生更完整和准确的基因组，使令人望而生畏的基因组从头测序成为可能，并使个人基因组重新测序和元基因组学变得容易处理。

DESCRIPTION (provided by applicant): Next-generation sequencing (NGS) platforms are fundamentally altering genetic and genomic research by providing massive amounts of data in a low-cost, high-throughput format. The main drawback of existing technologies is the short sequence read lengths they produce. Existing library prep methods are also constrained in producing short inserts of only a few kb. As a result, de novo assembly of genomes is not practical with short read NGS technologies alone. Even with a high quality reference human genome, resequencing and assembly of new human genomes is a significant challenge when analyzing complex genomic regions. Haplotyping across more than a few kb is not achieved without resorting to cloned DNA. New tools that bridge the gap between massively parallel short read sequencing technologies (<1,500 bases) and the need for large scaffolds > 20 kb to assemble a genome are clearly needed. The SBIR Phase I grant proposal "New Tools for Structural Variation Analysis, De Novo Assembly and Closing of Complex Genomes" proposes to develop a new "front end" to NGS and the software to support it. The technology to construct clone-free 20-40 kb mate pair libraries from large randomly sheared DNA fragments does not yet exist. This technology will enable the accurate assembly of complex genomes, much like fosmid and BAC end sequences in conventional clone based strategies. The development of these tools could reduce manual closing costs and computational costs of genome assembly by orders of magnitude produce more complete and accurate genomes, enable the de novo sequencing of daunting genomes, and make personal genome resequencing and metagenomics tractable.