EAGER: PLATE-seq: Development and Optimization of a New, Massively Parallel Sequencing Technology to Enable the Construction of a Fully-Sequenced Single-Colony Rice ORFeome

随着下一代测序技术的快速发展，目前已有80多种不同植物的基因组序列，其中一个物种中的数百至数千株植物目前正在进行测序。虽然基因组序列提供了每个物种中数以万计的蛋白质编码基因的部件清单，但大多数植物物种中的绝大多数基因仍有待功能注释，这已成为整个植物生物学领域的一大瓶颈。在全基因组范围内确定蛋白质功能的任何努力的先决条件是构建一组细菌克隆或文库，其中插入的片段仅代表基因的蛋白质编码区或开放阅读框架(ORF)。这些库通常被称为ORFeome。构建ORFeome文库的一个重大障碍是必须对数万个克隆进行单独测序，这使得这一过程非常劳动密集型和成本高昂，因为下一代测序(NGS)技术不能直接应用。这个迫切的项目旨在开发一种大规模并行测序技术，称为PLATE-SEQ(聚合酶链接法条形码适配器与核酸元件进行测序)，它将极大地提高产量并降低大规模测序工作的成本。作为概念验证的一部分，并展示盘子序列的实用性，该项目将构建第一个完全测序的单一群体水稻(Oryza sativa，cv.日本晴)或拥有约3,000个基因的基因组文库。这个渴望的项目本质上是跨学科的，代表着首席研究员在职业生涯中期将研究兴趣重新定位到植物科学领域，并将为科学、技术、工程和数学(STEM)专业的本科生和研究生提供研究培训。所有议定书都将通过出版物、研讨会和培训讲习班获得。此外，所有与该项目相关的ORF克隆、大肠杆菌和酵母菌株、计算工具和序列数据都将公开提供。下一代测序技术需要将数万个样本混合在一起进行集体测序。多重策略为跟踪单个样本的需要提供了部分解决方案，但当需要将序列与数千个单独样本进行匹配时，成本可能高得令人望而却步，这就是构建ORFeome文库的情况。因此，大规模的桑格测序虽然非常昂贵，但对于许多这样的应用来说仍然是必要的。PLATE-SEQ平台基于一种创新但未经验证的设计，该设计需要来自数千个大肠杆菌或酵母菌落的大规模嵌套缝合PCR，其中一个步骤中使用~150bP双链DNA作为引物会显著降低PCR效率。如果成功，平板序列技术将完全取代大规模Sanger测序的需要，预计将使ORFeome文库的测序效率比现有的多重NGS方法提高约1000倍，与传统的Sanger测序相比更是如此。因此，平板序列不仅对构建ORFeome文库具有巨大的意义，而且对于许多功能基因组学和反向蛋白质组学应用也具有巨大的意义，在这些应用中，测序读数必须追溯到单个样本，或者需要跟踪样本之间的关联，例如在酵母双杂交(Y2H)或其他选择和鉴定DNA分子对的遗传筛选中。

With the rapid development of next-generation sequencing technologies, the genome sequences of over 80 different plant species are available with several hundreds to thousands of individual plants within a species currently being sequenced. While the genome sequences provide the "parts lists" for tens of thousands of protein-coding genes in each species, the vast majority of genes in most plant species remain to be functionally annotated, which has become a major bottleneck for the whole plant biology field. A prerequisite for any effort to determine the functions of proteins on a genome-wide scale requires the construction of a set or library of bacterial clones with inserts representing only the protein coding regions or open reading frames (ORFs) of genes. These libraries are often referred to as an ORFeome. A significant hurdle for constructing an ORFeome library is that tens of thousands of clones must be sequenced individually, making the process extremely labor-intensive and cost-prohibitive since next-generation sequencing (NGS) technologies cannot be applied directly. This EAGER project aims to develop a massively parallel sequencing technology, called PLATE-seq (PCR mediated linkage of barcoded adapters to nucleic acid elements for sequencing), which will drastically increase the throughput and reduce the cost of large-scale sequencing efforts. As part of the proof-of-concept and to demonstrate the utility of PLATE-seq, the project will construct the first fully-sequenced single-colony rice (Oryza sativa, cv. Nipponbare) ORFeome library for ~3,000 genes. This EAGER project is interdisciplinary in nature and represents a mid-career reorientation of research interest into the field of plant science for the Principal Investigator, and will provide research training for undergraduate and graduate students in Science, Technology, Engineering and Mathematics (STEM) majors. All protocols will be accessible through publications, seminars, and training workshops. In addition, all ORF clones, E. coli and yeast strains, computational tools, and sequence data generated in association with this project will be made openly available.Next-generation sequencing technologies require mixing tens of thousands of samples together to be sequenced en masse. Multiplexing strategies offer a partial solution to the need to track individual samples, but can be prohibitively expensive when sequences need to be matched to thousands of individual samples, which is the case for constructing an ORFeome library. For this reason, large-scale Sanger sequencing, albeit extremely expensive, is still necessary for many such applications. The PLATE-seq platform is based on an innovative but unproven design which requires large-scale nested stitch PCRs from thousands of E. coli or yeast colonies where the use of ~150 bp double-stranded DNA as a primer in one of the steps can significantly decrease the PCR efficiency. If successful, PLATE-seq technology will completely replace the need for large-scale Sanger sequencing, predicted to improve sequencing efficiency of ORFeome libraries ~1,000-fold over existing multiplexing NGS approaches, and even more so compared to traditional Sanger sequencing. For this reason, PLATE-seq has tremendous implications not only for constructing ORFeome libraries, but also for many functional genomics and reverse proteomics applications where it is essential that sequencing reads be traced back to individual samples, or where associations between samples need to be tracked such as in yeast two-hybrid (Y2H) or other genetic screens where pairs of DNA molecules are selected and identified.