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您提供关键见解 微生物群在炎症性肠病（IBD）中的功能。与腔和 粪便样品，粘膜相关的微生物组被认为与宿主更直接相关 免疫反应和疾病状态。但是，16S分析不允许低级分类学 功能电位和粘膜相关的微生物群的推断或表征 由于过于敏锐而不适合传统的全元基因组测序 高宿主DNA。迫切需要开发新的测序和分析 可以实现组织相关微生物群的无偏元基因组测序的方法 复杂的宿主 - 微生物系统。我将使用一个新颖的宿主元素元素 定义粘膜之间的组成和功能差异的测序方法， 腔和粪便微生物群，以及健康和疾病状态之间的前所未有的细节。 我的长期目标是为计算基因组学建立独立的研究计划 人类疾病和个性化医学专注于肠道微生物组和 IBD。该提议的中心假设是 粘膜相关的微生物群将识别特异性，物种和应变水平 与肠道炎症和人类疾病相关的组成和功能变异 发病。 我最近开发了一种新颖的测序和信息学协议，该协议与 现有的纳米孔测序技术可以使物种的动态选择和识别 或来自宏基因组样品的基因。这种方法可用于动态过滤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 应用此方法执行与粘膜相关的第一个有效的高深度shot弹枪测序 IL10 - / - 和野生型小鼠的回肠和结肠中的微生物群。使用这些数据，我 将比较主机耗尽的深度测序与传统的短读shot弹枪测序 和16S rRNA测序，用于分析依从性群落的组成和功能。我 将确定与结肠炎相关的分类学和遗传丰度的相对差异 小鼠模型，包括未被捕获的物种和应变水平变体 现有方法。我还将使用这种方法来确定是否依从性侵入性 大肠杆菌（AIEC）选择性地定居于粘膜表面相对于无菌的管腔 IL10 - / - 小鼠，支持它们作为小鼠模型中因果促炎剂的作用 结肠炎。最后，我将评估结肠中与粘膜相关的微生物组的变化 来自IBD和非IBD患者的活检样本以表征疾病行为 表型，有可能导致新颖的诊断和治疗工具。