A Web-Enabled Database for Rapid Metagenomic Biocatalyst Discovery and Validation

 描述(由申请人提供)：Radiant Genology建议开发一种集成的酶发现服务，酶变异引擎(EVE)，建立在迄今报道的最大克隆元基因组序列收集的基础上。其目标是将可公开访问的搜索引擎、注释丰富的序列数据库、排列的样本库和LIMS自动化平台相结合，以比DNA合成等替代选择更低的成本、更少的时间和更大的生物多样性池向最终用户提供新型酶变体。重要的是，这项服务克服了酶发现中的一个主要瓶颈，即传统上专注于容易培养的生物，现在已知这些生物在生物多样性中所占比例不到1%。达到或超过了第一阶段的研究和开发里程碑。特别是，我们成功地展示了一种高效的测序工作流程，它将允许我们对克隆文库进行测序和组装，预计克隆文库将编码约6亿个基因，其中99%来自未培养和基本上未研究的生物体。接下来，我们展示了一种组合条形码策略，它产生平均长度为30千碱基的组件，极大地改善了元基因组的邻接性。这一特征使人们能够发现功能相关的基因簇，例如那些编码复杂天然产物和营养固定的基因。这些服务被成功地整合到一个在线搜索引擎和电子商务平台中，网址为www.eve.bio。最后，我们开发和展示了一个自动化的LIMS基因恢复系统的基础设施，该系统每周可以从我们的阵列文库中恢复数千个感兴趣的基因。与第一阶段研究的成功相辅相成的是测序成本效率和云计算方面的全面改进。EVE服务已经获得了商业吸引力，我们相信进一步的发展将有利于基础研究，同时对广泛的生物制造过程产生积极影响。根据客户的反馈，这项建议的目标是1)使用邻接性保持策略继续对文库进行排序，2)计算基础设施的规模，3)开发先进的酶选择器和4)第三方数据库集成。该计划的总体结果将是一个集中化的搜索引擎，允许最终用户快速选择和接收生物信息学分析中识别的基因。与现有服务相比，这些基因将以更低的成本、更少的时间从更大的遗传多样性池中获得。总体而言，我们相信，我们的平台将提高我们对元基因组环境的序列与功能关系和注释的理解，帮助弥合尚未探索的遗传多样性池中的电子和生化特征之间的差距。

 DESCRIPTION (provided by applicant): Radiant Genomics proposes to develop an integrated enzyme discovery service, the Enzyme Variant Engine (EVE), built upon the largest cloned metagenomic sequence collection reported to date. The goal is to combine a publicly­accessible search engine, richly­annotated sequence database, arrayed sample library, and LIMS automation platform to deliver novel enzyme variants to end­users for lower cost, in less time, and from a greater pool of biodiversity than alternative options, such as DNA synthesis. Importantly, this service overcomes a major bottleneck in enzyme discovery that has traditionally focused on easily­cultivated organisms which are now known to represent less than 1% of biodiversity. Phase I research and development milestones were met or exceeded. In particular, we successfully demonstrated a high­efficiency sequencing workflow that will allow us to sequence and assemble our clone library, which is predicted to encode ~600M genes, >99% of which are derived from uncultivated and essentially unstudied organisms. We next demonstrated a combinatorial barcoding strategy that yields assemblies with an average length of >30 kilobases, a dramatic improvement in metagenomic contiguity. This feature enables the discovery of clusters of functionally related genes, such as those that encode complex natural products and nutrient fixation. These services were successfully integrated into an online search engine and e­commerce platform available at www.eve.bio. Finally, we developed and demonstrated infrastructure for an automated LIMS gene recovery system that can recover thousands of genes of interest from our arrayed library per week. The success of Phase I research was complemented by general improvements in sequencing cost­efficiency and cloud­computing. The EVE service has gained commercial traction and we believe further development will benefit basic research while positively impacting a broad range of biomanufacturing processes. Based on customer feedback, the aims of this proposal are 1) continued sequencing of the library using contiguity­preserving strategies 2) scaling of computational infrastructure 3) development of advanced enzyme selectors and 4) third­party database integration. The overall outcome of this program will be a centralized search engine which allows end­users to rapidly select and receive genes identified in bioinformatic analyses. These genes will be accessible for lower cost, in less time, and from a greater pool of genetic diversity than existing services. Overall, we believe that our platform will improve our understanding of sequence­to­function relationships and annotation for metagenomic environments, helping to bridge the gap between in silico and biochemical characterization from unexplored pools of genetic diversity.