Remnant diagnostic samples for high resolution genotyping of Chlamydia trachomatis

总结 人类沙眼衣原体感染可导致眼部和生殖道疾病，影响数百万人 每年的人。我们对C.沙眼的生物学是复杂的，其专性细胞内 自然和一个发展中的，但有限的遗传工具包。由于C. 在患者中发现的沙眼菌株中，下一代测序已经发展成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. 在患者人群中传播的沙眼病毒分离株。这种监测受到缺乏 最近培养的菌株，随着诊所完全转向核酸， 扩增试验用于衣原体诊断。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，用于评估当代应变变异性， 剩余临床样本作为材料来源。对所有已发表的C.沙眼衣原体 Genomes已经鉴定了一个大的候选寡核苷酸引物库， 基因组所有可变区测序的材料，我们已经证明， 诊断材料适用于基于PCR的方法。草案分析表明，所有的插入缺失和超过一半的 所有非保守性碱基变化中的一个可以在一组96个单独的PCR反应中评估。目标1： 将仔细评估不同的引物对，以确定一个集合，将涵盖最大部分的 以基于单个托盘的扩增形式扩增基因组。在目标2中，我们将在三个方面测试我们确定的引物集。 越来越复杂的基于机器人的分析，以评估系统的效用， 在一个单一的高通量实验中的遗传变异。该项目的最终产品将是一个灵活的 这一过程可以扩增和测序大量标本，将需要大约一周的时间 完成。我们预计，该平台将是有用的基础研究人员和临床科学家 探索衣原体基因型和临床表现之间的联系，并将作为一个模型， 对于感染诊断不适用的其他挑战性微生物， 包括培养步骤。

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.