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

对肠道微生物组的元基因组分析继续提供对 微生物区系在炎症性肠病中的作用。与Lumina和 粪便样本，与粘膜相关的微生物群被认为与宿主更直接相关 免疫反应和疾病状态。然而，16S图谱不允许低级别的分类学 功能潜能和粘膜相关微生物区系的推断或表征 不适用于传统的全基因组测序，因为 宿主DNA很高。迫切需要开发新的测序和分析 实现组织相关微生物区系无偏元基因组测序的方法 复杂的寄主-微生物系统。我将使用一种新的宿主耗尽的元基因组 用测序方法来确定粘膜、 鲁米纳和粪便微生物区系，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计划 人类疾病和个性化医学专注于肠道微生物组和 IBD。这一提议的中心假设是，元基因组测序 与粘膜相关的微生物区系将识别特定位置、物种和菌株水平 与肠道炎症和人类疾病相关的成分和功能变异 发病机制。 我最近开发了一种新的测序和信息学协议，它与 现有的纳米孔测序技术，能够动态选择和识别物种 或者来自元基因组样本的基因。这种方法可以用来动态过滤掉DNA 属于以前观察到的微生物物种或污染宿主基因组的序列。这就做 应用该方法对黏膜相关基因进行首次有效的高深度鸟枪法测序 IL10-/-和野生型小鼠回肠和结肠中的微生物区系。使用这些数据，我 将宿主耗尽的深度测序与传统的短读鸟枪测序进行比较 16S rRNA测序用于分析黏附群落的组成和功能。我 将确定与结肠炎相关的分类和基因丰度的相对差异 小鼠模型，包括未被捕获的物种和品系级别的变体 现有的方法。我还将使用这种方法来确定粘附性-侵入性 大肠埃希氏菌(AIEC)在无菌条件下，相对于肠腔选择性地定植于粘膜表面 IL10-/-小鼠，支持其在小鼠模型中作为因果促炎因子的作用 结肠炎。最后，我将评估结肠中与粘膜相关的微生物群的变化。 来自IBD和非IBD患者的活检样本以表征疾病行为 表型，有可能导致新的诊断和治疗工具。

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.