New Strategies for De Novo Sequencing of Daunting 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。因此，重新组装令人望而生畏的基因组仍然是不可能的，在分析复杂的基因组区域时，重新测序和组装人类基因组是一项重大挑战。显然需要新的工具来弥合大规模平行短读测序技术（35-500个碱基）和大型支架组装基因组（100,000个碱基）之间的差距。SBIR第一期拨款提案“令人望而生畏的基因组从头开始测序的新战略”建议开发一个新的NGS“前端”。从大的随机剪切DNA片段（50- 300kb）中构建无配对末端克隆文库的技术尚未发展起来。构建无克隆文库的高效通用协议将从50、100和300 Kb插入片段的配对末端生成长物理支架，从而实现复杂基因组的精确组装，就像传统克隆策略中的fosmid和BAC端序列一样。一种新的“虚拟BAC”图书馆建设技术将取代传统的基于克隆的方法。利用本文首次开发的新工具，将构建一个无克隆的300 Kb插入文库，并对单个成员进行完全测序。从染色体中产生大量连续的300 Kb序列区域，将极大地简化复杂基因组区域和复杂基因组的精确组装，就像传统策略中对整个BACs克隆的测序一样。这些工具的发展可以将基因组组装的计算成本降低2-3个数量级，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变得容易处理。1

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. As a result, de novo assembly of daunting genomes is still impossible and resequencing and assembly of human genomes is a significant challenge when analyzing complex genomic regions. New tools that bridge the gap between massively parallel short read sequencing technologies (35-500 bases) and the need for large scaffolds to assemble a genome (100,000 bases) are clearly needed. The SBIR Phase I grant proposal "New Strategies for De Novo Sequencing of Daunting Genomes" proposes to develop a new "front end" to NGS. The technology to construct paired-end clone-free libraries from large randomly sheared DNA fragments (50-300 kb) has not been developed. A high efficiency universal protocol for making clone-free libraries will generate long physical scaffolds from the paired-ends of 50, 100 and 300 Kb inserts, enabling the accurate assembly of complex genomes, much like fosmid and BAC end sequences in conventional clone based strategies. A new "virtual BAC" library construction technology will replace the conventional clone based method. A clone-free 300 Kb insert library will be constructed and individual members will be completely sequenced using the new tools developed for the first time in this proposal. The production of numerous contiguous 300 Kb regions of sequence from a chromosome will dramatically simplify the accurate assembly of complex genomic regions as well as complex genomes, much like the sequencing of entire BACs clone in conventional strategies. The development of these tools could reduce computational cost of genome assembly by 2-3 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. 1 PUBLIC HEALTH RELEVANCE: The practical result of this work will be the accurate assembly of complex regions of the human genome associated with disease, as well the ability to assemble entire genomes using random sequencing strategies. DNA sequencing of individual human genomes can unlock the genetic basis of complex diseases and as such is important to our medical well being. Metagenomic analysis of hundreds of unique organisms that cannot be cultivated can unlock new metabolic pathways for small molecule drugs and other industry applications. True de novo sequencing of novel genomes of complex organisms can shed light on comparative genomics, the evolutionary history of life, and better understanding of all life styles on earth, which forms a web that humans need for survival.