Detection of minority variants and mixed infections in Mycobacterium tuberculosis by whole genome sequencing using a specific-DNA capture strategy: A pilot study

Detection of mixed Mycobacterium tuberculosis infections is essential, particularly when resistance mutations are present in minority bacterial populations and may affect patient´s evolution and treatment. Whole genome sequencing has extended the amount of key information available for the diagnosis of Mycobacterium tuberculosis. The relevance of having genomic information at diagnosis for early intervention requires carrying out whole genome sequencing directly on the clinical samples. However, few studies have been successful with this approach due to the low representation of MTB-DNA in sputa. In this study, we evaluated the ability of a strategy based on specific MTB-DNA enrichment to detect minority variants and mixed infections by whole genome sequencing on controlled mixtures of MTB-DNAs in spiked sputa. A pilot study was carried out with 12 samples containing 98% of a DNA pool from sputa of non-tuberculosis patients and 2% of MTB-DNA mixtures at different proportions. Our strategy allowed us to obtain sequences with a quality equivalent to those obtained from culture: 62.5X depth coverage and 95% breadth coverage (for at least 20X reads). Assessment of minority variant detection was carried out by manually directed analysis and allowed to identify heterozygous positions up to 95:5 ratio. The strategy also distinguishes automatically mixed infections up to a proportion of 90:10. This strategy efficiently captures MTB-DNA in a non-specific genetic background, allows detecting minority variants and mixed infections, and has proven to be a promising tool for performing whole genome sequencing directly on clinical samples.

混合结核分枝杆菌 (Mycobacterium tuberculosis) 感染的检测至关重要，尤其是当耐药突变存在于少数菌群体中时，这类突变可能会影响患者的病程转归与治疗方案。全基因组测序已为结核分枝杆菌的诊断拓展了可获取的关键信息量。在诊断阶段即获取基因组信息以实现早期干预的重要性，要求直接对临床样本开展全基因组测序。然而，由于痰液中结核分枝杆菌DNA (MTB-DNA) 占比极低，该方法的成功应用案例鲜有报道。本研究针对加标痰液中的结核分枝杆菌DNA对照混合物，采用基于特异性MTB-DNA富集的策略开展全基因组测序，评估其检测少数变异株与混合感染的能力。本研究开展了一项预实验：共纳入12份样本，其中98%为非结核患者痰液的DNA混合池，剩余2%为不同比例配比的MTB-DNA混合物。本策略所获序列的质量可与培养样本的测序结果相当：测序深度达62.5倍，覆盖广度达95%（要求至少20倍读长覆盖）。通过人工定向分析评估少数变异株的检测效果，结果显示本策略可识别比例低至95:5的杂合位点。该策略还可自动区分比例低至90:10的混合感染样本。本策略可在非特异性遗传背景下高效富集MTB-DNA，能够实现少数变异株与混合感染的检测，已被证实是直接针对临床样本开展全基因组测序的极具应用前景的工具。