Remnant diagnostic samples for high resolution genotyping of Chlamydia trachomatis

摘要 人类感染沙眼衣原体可引起眼部和生殖道疾病，影响数百万人 每年的人口数。我们对沙眼衣原体生物学的理解因其专性的细胞内而变得复杂 自然和一个正在开发但有限的遗传工具包。由于它们之间的高度保护。 在患者中发现沙眼衣原体菌株，下一代测序已演变为一种直接的分析工具 病原体的遗传变异性。我们实验室的目标是监测C。 沙眼衣原体分离株在患者群体中传播。这一监测受到以下因素的严重影响 最近培养的菌株，随着临床完全转向核酸，它们正变得越来越罕见 衣原体诊断的扩增试验。为了在当前的诊断环境中实现我们的目标，我们 建议开发一种基于机器人的方法来评估当代应变变异性，并确定 以残存的临床标本作为材料来源。所有已发表沙眼衣原体的生物信息学分析 基因组公司已经鉴定出一个大的候选寡核苷酸引物文库，可以用来产生 对基因组的所有可变区进行测序的材料，我们已经证明了 诊断材料适用于基于聚合酶链式反应的方法。草案分析表明，所有的Indels和超过一半的人 在所有非保守的碱基变化中，可以在一组96个单独的PCR反应中进行评估。在目标1中，我们 将仔细评估不同的引物对，以确定将覆盖最大比例的 以单托盘为基础的扩增形式。在目标2中，我们将在三个阶段中测试我们识别的引物集 日益复杂的基于机器人的分析，以评估该系统在检查全球 单次高通量实验中的遗传变异。该项目的最终产品将是一款灵活的 可以放大大量样本并对其进行测序的过程，大约需要一周时间 来完成。我们预计这个平台将对基础研究人员和临床科学家有用 探索衣原体基因分型与临床表现之间的联系，并将作为一个模型 将该方法用于其他具有挑战性的微生物，而感染的诊断不适用于这些微生物 包括文化步骤。

Summary Chlamydia trachomatis infections in humans cause diseases of the eye and genital tract that affect millions of people annually. Our understanding of C. trachomatis biology is complicated by its obligate intracellular nature and a developing, but limited genetic tool kit. Because of the high level of conservation among C. trachomatis strains found in patients, next-gen sequencing has evolved into a straightforward tool for analysis of genetic variability of the pathogen. Our laboratory has a goal of monitoring genomic changes in C. trachomatis isolates circulating in patient populations. This monitoring is significantly affected by the lack of recent cultured strains, which are becoming increasingly rare as clinics move completely to nucleic acid amplification testing for chlamydial diagnosis. To address our goal in the current diagnostics climate, we propose to develop a robotics-based approach for evaluating contemporary strain variability, with deidentified remnant clinical samples as a source of material. Bioinformatic analysis of all published C. trachomatis genomes has identified a large library of candidate oligonucleotide primers that can be used to generate material for sequencing all variable regions of the genome, and we have demonstrated that remnant diagnostic material is suitable for a PCR-based approach. Draft analyses indicate that all indels and over half of all nonconservative base changes can be assessed in a set of 96 individual PCR reactions. In Aim 1, we will carefully evaluate different primer pairs to identify a collection that will cover a maximum fraction of the genome in a single tray-based amplification format. In Aim 2, we will test our identified primer set in three increasingly complicated robotics-based assays to assess the utility of the system for examining global genetic variation in a single high-throughput experiment. The final product of this project will be a flexible process that can amplify and sequence large collections of specimens and will take approximately one week to complete. We anticipate that this platform will be useful to basic researchers and clinician scientists exploring the connections between chlamydial genotype and clinical presentation, and will serve as a model for employing the approach with other challenging microorganisms for which diagnosis of infection does not include a culture step.