Advanced sequencing as a novel diagnostic tool to discover strain-level variation and function of mucosal-adherent bacteria contributing to IBD

先进测序作为一种新型诊断工具，可发现导致 IBD 的粘膜粘附细菌的菌株水平变异和功能

• 批准号: 10001012
• 负责人: Jeremy R Wang
• 金额: $ 15.06万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10001012
• 关键词: 16S ribosomal RNA sequencing; Activities of Daily Living; Bacteria; Behavior; Benign; Biological; Biological Assay; Biopsy; Biopsy Specimen; Case-Control Studies; Cecum; Characteristics; Classification; Colitis; Colon; Communities; Complex; Computing Methodologies; Crohn' s disease; DNA; DNA Sequence; Data; Disease; Distal; Escherichia coli; Genes; Genetic; Genome; Genomics; Germ-Free; Goals; Human; Ileocolitis; Immune System Diseases; Immune response; Individual; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Informatics; Interleukin-10; Intestines; Location; Metagenomics; Methods; Modernization; Mucous Membrane; Mus; Pathogenesis; Pathway interactions; Patients; Population; Protocols documentation; Proxy; Research; Resected; Resolution; Ribosomal RNA; Role; Sampling; Shotgun Sequencing; Shotguns; Site; Surface; System; Taxonomy; Technology; Time; Tissues; Variant; Wild Type Mouse; bacterial community; base; behavioral phenotyping; clinically significant; cost; deep sequencing; expectation; experimental study; fecal microbiota; genomic signature; gut microbiome; host microbiome; human DNA; human disease; ileum; inflammatory disease of the intestine; insight; metagenome; metagenomic sequencing; microbial; microbiome; microbiome research; microbiota; mouse model; mucosal microbiota; nanopore; novel; novel diagnostics; novel therapeutics; personalized medicine; programs; sequencing platform; tool; translational research program

Metagenomic analysis of the gut microbiome continues to provide critical insights into the function of microbiota in inflammatory bowel diseases (IBD). In contrast to luminal and fecal samples, the mucosa-associated microbiome is thought to be more directly relevant to host immune response and disease state. However, 16S profiling does not permit low-level taxonomic inference or characterization of functional potential and mucosa-associated microbiota are not amenable to traditional whole- metagenome sequencing due to prohibitively high host DNA. There is a critical need to develop novel sequencing and analysis methods that enable unbiased metagenomic sequencing of tissue-associated microbiota in complex host-microbiome systems. I will use a novel host-depleted metagenome sequencing approach to define the compositional and functional differences between mucosal, luminal, and fecal microbiota, and between healthy and disease states in unprecedented detail. My long-term goal is to establish an independent research program in computational genomics for human disease and personalized medicine focused on the gut microbiome and IBD. The central hypothesis of this proposal is that metagenomic sequencing of mucosa-associated microbiota will identify location-specific, species- and strain-level composition and functional variation associated with intestinal inflammation and human disease pathogenesis. I recently developed a novel sequencing and informatics protocol that interfaces with existing nanopore sequencing technology to enable dynamic selection and identification of species or genes from a metagenomic sample. This approach can be used to dynamically filter out DNA sequences belonging to previously observed microbial species or contaminating host genome. I will apply this method to perform the first effective high-depth shotgun sequencing of mucosa-associated microbiota in the ileum and colon of Il10-/- and wild-type mice. Using these data, I will compare host-depleted deep sequencing to traditional short-read shotgun sequencing and 16S rRNA sequencing for assaying composition and function of adherent communities. I will identify relative differences in taxonomic and genic abundances associated with colitis in a mouse model, including species- and strain-level variants that are not captured by existing approaches. I will also use this approach to determine whether adherent-invasive Escherichia coli (AIEC) selectively colonize the mucosal surface relative to the lumen in germ-free Il10-/- mice, supporting their role as causal pro-inflammatory agent in a mouse model of colitis. Lastly, I will assess variation in the mucosa-associated microbiome in colon biopsy samples from IBD and non-IBD patients to characterize disease behavioral phenotypes, potentially leading to novel diagnostic and therapeutic tools.

肠道微生物组的宏基因组分析继续为以下方面提供重要见解： 微生物群在炎症性肠病（IBD）中的作用。与Luminal相比， 对于粪便样本，粘膜相关微生物组被认为与宿主更直接相关 免疫反应和疾病状态。然而，16 S分析不允许低水平的分类学 功能潜力和粘膜相关微生物群推断或表征 不适合传统的全宏基因组测序， 高宿主DNA。迫切需要开发新的测序和分析方法 本发明提供了能够对哺乳动物中的组织相关微生物群进行无偏宏基因组测序的方法， 复杂的宿主微生物系统。我会用一种新的宿主缺失宏基因组 测序方法来确定粘膜， 管腔和粪便微生物群，以及健康和疾病状态之间前所未有的细节。 我的长期目标是建立一个独立的计算基因组学研究项目， 人类疾病和个性化医疗集中在肠道微生物组， IBD。这一提议的中心假设是， 粘膜相关微生物群将确定特定位置，物种和菌株水平 与肠道炎症和人类疾病相关的组成和功能变化 发病机制 我最近开发了一种新的测序和信息学协议， 现有的纳米孔测序技术， 或来自宏基因组样本的基因。这种方法可以用来动态过滤出DNA 属于先前观察到的微生物物种或污染宿主基因组的序列。我会 应用这种方法进行第一次有效的高深度鸟枪测序粘膜相关的 图11显示了Il 10-/-和野生型小鼠的回肠和结肠中的微生物群的图。利用这些数据，我 将比较宿主耗尽的深度测序和传统的短读鸟枪测序 和16 S rRNA测序用于测定粘附群落的组成和功能。我 将确定与结肠炎相关的分类学和基因丰度的相对差异， 小鼠模型，包括未被捕获的种属和品系水平变异 现有的方法。我还将使用这种方法来确定是否有粘连-侵入性 大肠杆菌（Escherichia coli，AIEC）在无菌环境中选择性地定殖于相对于管腔的粘膜表面。 IL 10-/-小鼠，支持其在小鼠模型中作为致病促炎剂的作用 结肠炎最后，我将评估结肠粘膜相关微生物组的变化 来自IBD和非IBD患者的活检样品，以表征疾病行为 表型，可能导致新的诊断和治疗工具。