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。因此，令人望而生畏的基因组从头组装仍然是不可能的，而人类基因组的重新测序和组装在分析复杂的基因组区域时是一个巨大的挑战。显然需要新的工具来弥合大规模并行短读测序技术(35-500个碱基)和组装基因组所需的大型支架(100,000个碱基)之间的差距。SBIR第一阶段赠款提案“令人望而生畏的基因组去新测序的新战略”提议开发一个新的NGS“前端”。从大的随机剪切的DNA片段(50-300kb)中构建成对端无克隆文库的技术尚未开发出来。一种用于制作无克隆文库的高效通用协议将从50、100和300kb插入片段的配对末端生成长的物理支架，从而能够准确地组装复杂的基因组，就像传统的基于克隆的策略中的Fosmidd和BAC末端序列一样。一种新的“虚拟BAC”建库技术将取代传统的基于克隆的方法。将构建一个无克隆的300kb插入文库，并将使用本提案中首次开发的新工具对个别成员进行完全测序。从一条染色体上产生大量连续的300kb序列区域将极大地简化复杂基因组区域和复杂基因组的准确组装，就像传统策略中对整个BAC克隆进行测序一样。这些工具的开发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。1 公共卫生相关性：这项工作的实际结果将是准确组装与疾病相关的人类基因组的复杂区域，以及使用随机测序策略组装整个基因组的能力。人类个体基因组的DNA测序可以揭开复杂疾病的遗传基础，因此对我们的医疗健康很重要。对数百种无法培养的独特生物进行元基因组分析，可以为小分子药物和其他工业应用打开新的代谢途径。对复杂生物的新基因组进行真正的从头测序可以揭示比较基因组学、生命的进化史，以及更好地理解地球上所有的生命方式，这形成了人类生存所需的网络。

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.