A method for the culture-free discovery and host affiliation of novel viruses from metagenomic samples

摘要 在这一应用中，我们建议开发一种新的试剂盒和配套的分析软件平台，该平台 采用Hi-C技术，以用户友好的方式从后基因组样本中组装病毒基因组，并 将这些病毒与它们的微生物宿主联系起来。该产品能够在无培养条件下发现噬菌体 直接在混合微生物群落中定量测量它们的寄主范围。 噬菌体是感染细菌的病毒；它们通过捕食宿主来塑造微生物生态系统。 通过水平基因转移。它们也是传播抗微生物药物的重要媒介。 抵抗。尽管它们对微生物生物学有很大的影响，但只有一小部分噬菌体基因组 在公共数据库中出现的部分原因是很难分离其宿主不可培养的噬菌体 实验室环境。我们理解病毒生物学的能力的这种限制对新出现的 生物技术的应用包括粪便微生物区系移植和噬菌体疗法以及广泛的 对微生物学研究的影响。 我们建议开发一种新的计算方法与改进的实验方法相结合来 解卷病毒基因组并从整个微生物组样本中执行宿主归属，而无需培养。在… 这种方法的核心是利用邻近连接方法(Hi-C)，这种方法在物理上将DNA 存在于完整微生物体内的序列。这为病毒的接触提供了直接的物理证据。 基因组序列和寄主从属关系信息，这是任何其他方法都无法实现的。 这一直接到第二阶段提案的结果将是工具包和面向用户的计算的组合 这一平台将使复杂的下一代测序生物学家和测序 新手可以询问有关他们感兴趣的微生物群落中的病毒的重要问题。目标1侧重于 计算方法改进了从元基因组数据中恢复病毒基因组。AIM 2开发 增强病毒Hi-C化学的方法。这两个目标协同工作以增强数据 质量。最后，Aim 3通过一个门户网站开发了一个用于数据分析的计算平台 可以通过网络浏览器或下载的原始数据直接可视化，以供进一步探索。完工后 我们将开发一个一流的商业平台来发现病毒基因组并识别它们的宿主 来自复杂的微生物群落。

ABSTRACT In this application, we propose to develop a novel reagent kit and accompanying analytic software platform that employs Hi-C technology for a user-friendly method to assemble viral genomes from metagenomic samples and associate these viruses with their microbial hosts. This product enables culture-free discovery of phages and quantitative measurement of their host range directly in mixed microbial communities. Bacteriophages are viruses that infect bacteria; they shape microbial ecosystems through predation on hosts and through horizontal gene transfer. They are also an important vector in the transmission of anti-microbial resistance. Despite their potent impact on microbial biology, only a tiny proportion of phage genomes are represented in public databases in part because of the difficulty of isolating phage whose hosts are not culturable laboratory settings. This limitation in our ability to understand viral biology has important impacts on emerging biotechnology applications including fecal microbiota transplantation and phage therapy as well as wide-ranging effects on microbiological research. We propose to develop a novel computational approach combined with improved experimental methods to deconvolute viral genomes and perform host attribution from whole microbiome samples without culturing. At the core of this approach is utilization of proximity ligation methods (Hi-C) which physically associates DNA sequences present within intact microbes. This provides direct physical evidence of the contiguity of viral genome sequences and host affiliation information that is not achievable by any other method. The outcome of this direct-to-Phase II proposal would be a combination of kit and user-facing computational platform that would empower both the sophisticated next-generation-sequencing biologist and the sequencing novice to ask important questions about viruses in their microbial community of interest. Aim 1 focuses on computational methods improve the recovery of viral genomes from metagenomic data. Aim 2 develops methodologies to enhance viral Hi-C chemistry. These two aims work together synergistically enhance data quality. Finally, Aim 3 develops a computational platform for data analysis through a web portal where results could be visualized directly via web browser or raw data downloaded for further exploration. Upon completion we will have developed a first-in-class commercial platform to discover viral genomes and identify their hosts from complex microbial communities.