EAGER: Collaborative Research: Exploratory application of single-molecule real time (SMRT) DNA sequencing in microbial ecology research

微生物群落是维持生态系统养分平衡并最终塑造整个生态系统功能的全球生物地球化学循环的主要驱动力。今天，我们认识到微生物在营养元素的生化过程中发挥的关键作用；然而，我们对微生物群落结构如何影响给定营养周期内的一系列生物地球化学过程的理解有些初级。对微生物群落和生物地球化学循环之间的结构-功能关系的了解有限，这在很大程度上是由于在描述微生物群落生态特征方面的技术限制。直到最近，微生物生态学家还没有办法明确地描述微生物群落中物种的丰富度和均衡性，以及组成微生物种群的普遍生化能力。利用高通量DNA测序和三种方法学方法，鸟枪式元基因组学、聚合酶链式反应扩增序列和基因组学微生物生态学家开始揭示微生物群落的内部工作机制，并将遗传细节与生物地球化学过程联系起来。虽然这项工作为微生物生态学的发展带来了巨大的希望，但目前可用的高通量测序技术并不理想地适合生态系统科学的高样本吞吐量要求、细菌和病毒的小基因组尺寸以及它们的遗传新颖性。此外，在某些情况下，下一代DNA测序仪未能充分真实地应用于环境DNA样本，造成了数据偏见和错误的科学结论。这项“高风险、高回报”的研究旨在探索和测试新一代DNA测序仪--PacBio RS的使用，该测序仪具有几个属性，可能使其更适合微生物生态学研究的特定需求，并有可能对这一地球科学学科产生极大的变革。将进行一系列受控和仔细重复的实验，以测试PacBio测序在鸟枪式元基因组学、16S PCR扩增子测序和单细胞基因组测序中的使用。该项目将利用来自其他高通量测序平台(如Illumina和454)的现有数据集，直接比较PacBio在每个应用领域的性能。通过授予特拉华大学的NSF重大研究仪器奖，PI将获得学术机构提供的为数不多的PacBio遥感仪器之一。最终，这些研究将限制PacBio测序中的实验误差，并作为该仪器用于微生物生态学研究的初步示范。这项提议的更广泛影响包括努力了解和限制PacBio测序中的错误和其他偏差的来源，并提出技术建议，以确定该仪器在微生物科学中的最佳使用。在这项工作的过程中，PIS将指导一名博士研究生和一名博士后研究员，并提供对所有项目数据和发现的开放访问。

Microbial communities are the primary drivers of the global biogeochemical cycles that maintain the nutrient balance of ecosystems and ultimately shape overall ecosystem function. Today, we appreciate the critical role microbes play in the biochemical processing of nutrient elements; yet our understanding of how the structure of microbial communities influences the suite of biogeochemical processes within a given nutrient cycle is somewhat rudimentary. Limited understanding of structure-function relationships between microbial communities and biogeochemical cycles is due in large part to technological limitations in characterizing the ecology of microbial communities. Until recently, microbial ecologists simply had no means of unambiguously characterizing the richness and evenness of species within a microbial community and the prevailing biochemical capabilities of constituent microbial populations. Using high-throughput DNA sequencing and three methodological approaches,shotgun metagenomics, PCR amplicon sequencing, and genomics microbial ecologists are beginning to unveil the inner workings of microbial communities and connect genetic details with biogeochemical processes. While this work holds great promise for the advancement of microbial ecology, currently-available high-throughput sequencing technologies are not ideally suited to the high-sample throughput demands of ecosystem science, the small genome size of bacteria and viruses, and their genetic novelty. Moreover, in some cases the failure to adequately ground-truth application of next-generation DNA sequencers to environmental DNA samples has resulted in biased data and erroneous scientific conclusions. This "high-risk; high-reward" research seeks to explore and test the use of a new, next-generation DNA sequencer, the PacBio RS, which has several attributes that may make it better suited to the specific needs of microbial ecology research and has the potential to be highly transformative to this geoscience discipline. A series of controlled and carefully replicated experiments will be conducted that will test the use of PacBio sequencing for shotgun metagenomics, 16S PCR amplicon sequencing, and single cell genome sequencing. This project will leverage existing datasets from other high-throughput sequencing platforms (e.g., Illumina and 454) to directly compare the performance of PacBio in each of these application areas. Through a NSF Major Research Instrumentation award to the University of Delaware, the PIs will have access to one of the few PacBio RS instruments available at an academic institution. Ultimately, these investigations will constrain the experimental error within PacBio sequencing and serve as an initial demonstration of the utility of the instrument for microbial ecology research. The Broader Impacts of this proposal includes an effort to understand and constrain the sources of error and other biases within PacBio sequencing, and make technical recommendations that will shape the optimal use of the instrument within microbial science. In the course of this work, the PIs will mentor a Ph.D. graduate student and a post-doctoral researcher, and provide open access to all project data and findings.