A proximity ligation method to track mobile element hosts

摘要 在这个应用中，我们建议开发Hi-C技术和数据分析软件，以方便用户使用 抗菌素耐药(AMR)和含有毒力基因的移动元件与其相关的 细菌菌株。 可移动元件是编码功能元件的染色体外DNA或RNA分子。这些 分子可以在同一物种的成员之间和不同物种之间传播，导致 通过共享可移动元素编码的蛋白质，生化途径的快速进化。这有 对微生物群落进化的深刻影响，特别是对耐药性和 社区成员的毒力档案。 到目前为止，还没有一种经济有效的方法来识别混合微生物群落中的移动元素宿主： 很大一部分微生物在实验室环境和当前的鸟枪基因组下是无法培养的 方法无法捕获主机信息。因此，需要一种新的工具来提供关键的信息 了解移动元素生物学。染色体确认捕获(3C)和Hi-C等方法可以 用于快速、高精度地识别共存于单个细胞内的DNA分子 直接从混合培养物或生物样本中提取。这项提议的目标是开发一类新的工具 这可以用来识别复杂微生物群落中移动元素的宿主和传播。 在此SBIR第二阶段应用中提出的产品满足了快速、无培养鉴定的需求 复杂微生物群落中移动元素-宿主关系的研究。该方法建立在阶段的基础上 基因组学在使用Hi-C和3C技术方面的专业知识，这允许解卷积复杂的 微生物群落中的细胞，而不需要实验室培养。 在此应用程序中，我们提出了一种降低成本、提高速度并简化 分析以邻近连接为基础的试验，以将质粒与其宿主相关联。我们的方法基于两个 离散工作包： 目标1的重点是结合靶标浓缩和邻近连接方法来降低测序成本 与后基因组去卷积相关。在目标2中，我们将开发和设计一个面向客户的门户网站 用于上传和分析数据，我们的客户可以在其中轻松导航。 当拟议项目完成后，我们将为第一代目标浓缩奠定基础- C工具包和服务，这将使我们的创新平台商业化。

ABSTRACT In this application, we propose to develop Hi-C technology and data analysis software for a user-friendly method to associate antimicrobial resistant (AMR) and virulence gene-containing mobile elements with their associated bacterial strains. Mobile elements are extra-chromosomal DNA or RNA molecules that encode functional elements. These molecules can be transmitted between members of the same species and across disparate species, leading to the rapid evolution of biochemical pathways through the sharing of mobile element-encoded proteins. This has a profound influence on the evolution of microbial communities, with particular impact on the drug resistance and virulence profiles of the members of the communities. To date, there is no cost-effective method for identifying mobile element hosts in a mixed microbial community: A significant fraction of microorganisms are unculturable under laboratory settings and current shotgun genomic methods fail to capture host information. Therefore, a new tool is needed to provide the information critical to understanding mobile element biology. Methods such as chromosome confirmation capture (3C) and Hi-C can be used rapidly and with high accuracy to identify DNA molecules that co-exist within individual cells directly from a mixed culture or biological sample. The goal of this proposal to develop a new class of tools that can be used to identify the hosts and transmission of mobile elements in a complex microbial community. The product proposed in this SBIR Phase II application addresses the need for rapid, culture-free identification of mobile element-host relationships in complex microbial communities. The method is built upon Phase Genomics’ expertise with the use of Hi-C and 3C techniques, which allows to deconvolve complex mixtures of cells within microbial communities, without the need for laboratory culture. In this application, we propose to produce a method that reduces cost, increases speed, and simplifies the analysis of a proximity ligation-based test to associate plasmids with their host. Our approach is based on two discrete work packages: Aim 1 is focused on combining target enrichment and proximity-ligation methods to reduce sequencing costs associated with metagenomic deconvolution. In Aim 2, we will develop and design a customer-facing web portal for upload and analysis of data in which our clients can navigate easily. Upon completion of the proposed project, we will have laid the basis for a first-generation target enrichment Hi- C kit and service that will make our innovative platform commercially available.