A proximity ligation method to track mobile element hosts

摘要 在此应用中，我们提出了开发Hi-C技术和数据分析软件的用户友好的方法 将含抗菌素耐药（AMR）和毒力基因的移动的元件与其相关的 细菌菌株。 移动的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传播，也可以在不同的物种之间传播， 通过共享移动的元件编码的蛋白质，生物化学途径的快速进化。这 对微生物群落的进化产生深远的影响，特别是对耐药性的影响， 社区成员的毒力特征。 到目前为止，还没有一种经济有效的方法来鉴定混合微生物群落中的移动的元素宿主： 很大一部分微生物在实验室环境下是不可培养的，并且目前的鸟枪基因组技术不能培养微生物。 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信息， 了解移动的元素生物学。染色体确认捕获（3C）和Hi-C等方法可以 快速且高精度地用于鉴定单个细胞内共存的DNA分子 直接来自混合培养物或生物样品。该提案的目标是开发一类新的工具， 可以用来识别宿主和复杂微生物群落中移动的元素的传播。 SBIR II期申请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的元素-宿主关系。该方法基于阶段 Genomics在使用Hi-C和3C技术方面的专业知识，可以对复杂的混合物进行解卷积， 细胞内的微生物群落，而不需要实验室培养。 在本申请中，我们提出了一种方法，该方法降低了成本，提高了速度，并简化了计算。 分析将质粒与其宿主相关联的基于邻位连接的测试。我们的方法基于两个 离散工作包： 目标1集中于结合靶富集和邻位连接方法以降低测序成本 与宏基因组去卷积相关。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，我们的客户可以轻松浏览。 在拟议项目完成后，我们将为第一代目标浓缩奠定基础。 C套件和服务，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.