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、高通量的形式提供大量数据，从根本上改变了遗传和基因组研究。现有技术的主要缺点是它们产生的序列读取长度较短。现有的库准备方法在生成只有几kb的短插入时也受到限制。因此，单靠短读NGS技术进行基因组从头组装是不现实的。即使有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是一个重大挑战。如果不借助克隆DNA，超过几kb的单倍型是无法实现的。我们显然需要新的工具来弥补大规模平行短读测序技术（< 1500个碱基）与大型支架（bbb20 kb）之间的差距。SBIR第一期拨款提案“结构变异分析，从头组装和关闭复杂基因组的新工具”建议开发一个新的NGS“前端”和支持它的软件。从大量随机剪切的DNA片段中构建无克隆的20- 40kb配对文库的技术尚不存在。这项技术将使复杂基因组的精确组装成为可能，就像传统克隆策略中的fosmid和BAC端序列一样。这些工具的发展可以在数量级上降低人工关闭成本和基因组组装的计算成本，产生更完整和准确的基因组，使艰巨的基因组重新测序成为可能，并使个人基因组重测序和宏基因组学易于处理。

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.