Microbial communities are inter-connected systems of incredible complexity and dynamism that play crucial roles in health, energy, and the environment. To better understand microbial communities and how they respond to change, it is important to know which microbes are present and their relative abundances at the greatest taxonomic resolution possible. Here, we describe a novel protocol (RoC-ITS) that uses the single-molecule Nanopore sequencing platform to assay the composition of microbial communities in unprecedented detail. This methodology produces long-read sequences including multiple copies of the same complete 16S ribosomal gene and its neighboring internally transcribed spacer (ITS) using rolling-circle amplification. The ribosomal 16S gene provides phylogenetic information down to the species-level, while the much less conserved ITS region contains strain-level information. When linked together, this combination of markers allows for the identification of individual ribosomal units within a specific organism, the assessment of their relative stoichiometry, and the ability to monitor subtle shifts in microbial community composition with a single generic assay. We applied RoC-ITS to a mock microbial community that was also sequenced using the Illumina platform, demonstrating its accuracy in quantifying the relative abundance and identity of each species.

中文翻译：

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RoC-ITS对微生物群落的高分辨率系统发育和种群遗传分析

微生物群落是相互联系的系统，具有难以置信的复杂性和活力，在健康，能源和环境中发挥着至关重要的作用。为了更好地了解微生物群落及其对变化的响应，重要的是要知道可能存在的微生物及其相对丰度，且可能以最大的分类学分辨率。在这里，我们描述了一种新颖的协议（RoC-ITS），该协议使用单分子纳米孔测序平台以前所未有的细节分析了微生物群落的组成。该方法使用滚环扩增法产生了包含同一完整16S核糖体基因及其相邻内部转录间隔子（ITS）的多个拷贝的长读序列。核糖体16S基因提供了直至物种水平的系统发育信息，而保守程度较低的ITS区域则包含应变级别的信息。当链接在一起时，这种标记组合可以识别特定生物体中的单个核糖体单位，评估其相对化学计量，并能够通过单个通用测定监测微生物群落组成的细微变化。我们将RoC-ITS应用于模拟微生物群落，该群落也使用Illumina平台进行了测序，证明了其在定量每种物种的相对丰度和同一性方面的准确性。并具有通过单一通用测定监测微生物群落组成细微变化的能力。我们将RoC-ITS应用于模拟微生物群落，该群落也使用Illumina平台进行了测序，证明了其在定量每种物种的相对丰度和同一性方面的准确性。并具有通过单一通用测定监测微生物群落组成细微变化的能力。我们将RoC-ITS应用于模拟微生物群落，该群落也使用Illumina平台进行了测序，证明了其在定量每种物种的相对丰度和同一性方面的准确性。