EAGER: Development of a tool to rapidly and cost-effectively sequence the exome of any organism

了解基因型、表型和环境之间的相互作用是生物学中最大的挑战之一。研究人员在实验设计中面临着双重挑战：1)对足够多的个体进行采样，以准确地描述种群特征；2)对基因组中最具信息量的部分（导致表型变化的碱基对）进行测序。即使在DNA测序方面取得了重大技术进步，但对大多数生物体来说，对多个个体的整个基因组进行测序仍然过于昂贵。利用探针靶向基因组中的特定位置具有将测序集中在基因组功能区域的优势。然而，目前的方法需要预先存在的基因组资源来设计探针和大量的财政资源来合成探针，限制了对已经充分研究的生物体和长期项目的使用。这个冒险的项目将开发一种方法来捕获基因组的特定区域进行测序，而不需要现有的基因组资源，从而消除了探针开发的时间和成本。这种新方法将能够对参与适应的基因组部分进行快速和经济的测序，并将提供前所未有的能力来检测圈养和野生种群的选择。如果成功，这种新方法将能够评估对短期生态灾害和长期气候变化的快速适应，直接协助成功的缓解、保护和恢复工作。提出的研究的首要目标是将表达外显子组捕获测序（EecSeq）发展成为一种成本和时间效率高的任何生物体外显子组捕获方法。为此，该项目采取三个阶段的方法，(1)专注于实验室方案的优化和改进，(2)利用全基因组测序（WGS）、传统外显子组捕获和RNAseq进行验证，以及(3)开发开源、可重复的生物信息学管道，包括从头组装。第一阶段将优化EecSeq协议中的三个关键要素：探针和插入长度、探针和捕获池多样性以及杂交过程。优化这三个参数将最大限度地增加测序的外显子组碱基对和靶reads的数量，增加可以同时测序的个体数量，同时大大降低成本。该项目的第二阶段将使用两种独立的方法来验证EecSeq基因型，包括将EecSeq与传统的外显子组捕获进行比较，一组具有WGS数据的参考个体。最后阶段将利用东部牡蛎基因组的染色体水平组装以及前两个阶段的结果，为EecSeq开发两种互补的从头组装方法：一种利用捕获的基因组读取，一种混合方法将利用cDNA探针（测序时）和捕获的基因组读取的序列。所有的实验、数据分析和演示都将在一个完全开放和可重复的科学管道中进行，这将导致一个有效的一步一步的实验室协议和一个全新的EecSeq生物信息学管道，该管道包括任何生物体的基因座组装和注释。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Understanding the interaction between genotype, phenotype, and the environment is one of the greatest challenges in biology. Researchers face a two-fold challenge in experimental design: 1) sampling enough individuals to accurately characterize populations and 2) sequencing the most informative part of the genome, the base pairs that cause phenotypic change. Even with major technological advances in DNA sequencing, it is still too expensive, for most organisms, to sequence the entire genome of multiple individuals. Using probes to target specific locations in the genome has the advantage of focusing sequencing on the functional areas of the genome. However, current methods require preexisting genomic resources for probe design and substantial financial resources for probe synthesis, limiting use to already well-studied organisms and long-term projects. This risky project will develop a method for capturing specific areas of the genome for sequencing with no need for existing genomic resources, removing the time and cost of probe development. This new method will enable the rapid and cost-effective sequencing of the portions of the genome involved in adaptation and will provide unprecedented capacity to detect selection in captive and wild populations. If successful, this new method will enable the assessment of rapid adaptation to short-term ecological disasters and long-term climate change, directly assisting with successful mitigation, conservation, and restoration efforts.The over-arching goal of the proposed research is to develop Expressed Exome Capture Sequencing (EecSeq) into a cost and time efficient method of exome capture for any organism. To do this, the project takes a three phased approach, (1) focusing on laboratory protocol optimization and improvement, (2) validation with whole genome sequencing (WGS), traditional exome capture, and RNAseq, and (3) the development of an open source, reproducible bioinformatics pipeline, including de novo assembly. Phase one will optimize three key elements in the EecSeq protocol: probe and insert length, probe and capture pool diversity, and the hybridization process. Optimizing all three parameters will maximize the number of sequenced exomic basepairs and on-target reads, increasing the number of individuals that can be sequenced simultaneously while greatly reducing costs. The second phase of the project will use two independent approaches to validate EecSeq genotypes, including a comparison of EecSeq to traditional exome capture, a set of reference individuals with WGS data. The final phase will leverage the chromosome-level assembly of the eastern oyster genome along with results from both previous phases to develop two complementing de novo assembly methods for EecSeq: one utilizing captured genomic reads and a hybrid method that will utilize sequences from the cDNA probes (when sequenced) and captured genomic reads. All experiments, data analysis, and presentation will take place in a completely open and reproducible science pipeline, which should lead to an efficient step-by-step laboratory protocol and a de novo bioinformatic pipeline for EecSeq that incorporates locus assembly and annotation for any organism.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.