Detection of low-level mixed-population drug resistance in Mycobacterium tuberculosis using high fidelity amplicon sequencing

The study''s goal was to describe the method of “single molecule-overlapping reads” (SMOR) analysis with next generation ligation-free amplicon sequencing for determination of ultra-rare target alleles in complex mixtures that provides an increased sensitivity over standard DNA sequencing. The method was described using standardized mixtures of DNA from resistant and susceptible Mycobacterium tuberculosis isolates and the assay’s performance for detecting ultra-rare drug resistance subpopulations in DNA extracted directly from clinical sputum samples.

本研究旨在阐明「单分子重叠读段（single molecule-overlapping reads，SMOR）」分析法结合下一代无连接子扩增子测序（next generation ligation-free amplicon sequencing）的技术流程，用于复杂混合样本中超稀有靶等位基因的检测；该方法相较标准DNA测序具有更高的检测灵敏度。本研究采用耐药与易感结核分枝杆菌（Mycobacterium tuberculosis）分离株的标准化DNA混合样本对该方法进行了阐述，并评估了该检测方法在直接从临床痰液样本中提取的DNA内检测超稀有耐药亚群的性能。