Optimization of Mycobacterium tuberculosis DNA processing prior to whole genome sequencing

IF 2.8 3区医学 Q3 IMMUNOLOGY

Tuberculosis Pub Date : 2024-07-11 DOI:10.1016/j.tube.2024.102543

Samira Dziri , Julie Marin , Pauline Quagliaro , Charlotte Genestet , Oana Dumitrescu , Etienne Carbonnelle , Typhaine Billard-Pomares

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Abstract

The process of whole genome sequencing of the Mycobacterium tuberculosis complex is dependent on complete the inactivation of the strain and subsequent DNA extraction. The objective of this study was to optimise the two steps.

Firstly, the efficacy of Triton X-100 as a solvent for the inactivation step was evaluated. This solvent has been demonstrated to be effective in killing bacteria and is less toxic than the previously employed chloroform. For the extraction step, two lysis methods were evaluated: enzymatic (B1 protocol) and mechanical (B2 protocol). For whole genome sequencing, the Nextera XT DNA library preparation protocol was performed for both the B1 and B2 protocols. Subsequently, each library was subjected to whole-genome sequencing. The results demonstrated that heat lysis inactivation with Triton was effective, with no bacteria remaining viable following this treatment. The enzymatic and mechanical extraction protocols yielded comparable results in terms of DNA quantity and quality. The sequencing results showed that there was no significant difference in read depths between the two protocols. In conclusion, for MTBC strains, we recommend the use of our Triton inactivation method, which meets biosafety expectations.

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优化全基因组测序前的结核分枝杆菌 DNA 处理。

结核分枝杆菌复合体的全基因组测序过程取决于菌株的完全灭活和随后的 DNA 提取。本研究的目的是优化这两个步骤。首先，评估了 Triton X-100 作为灭活步骤溶剂的功效。这种溶剂已被证明能有效杀死细菌，而且比以前使用的氯仿毒性更低。在提取步骤中，评估了两种裂解方法：酶法（B1 方案）和机械法（B2 方案）。对于全基因组测序，B1 和 B2 方案均采用 Nextera XT DNA 文库制备方案。随后，对每个文库进行全基因组测序。结果表明，用 Triton 进行热裂解灭活是有效的，处理后没有细菌存活。酶法和机械提取法在 DNA 数量和质量方面的结果相当。测序结果显示，两种方案的读取深度没有明显差异。总之，对于 MTBC 菌株，我们建议使用我们的 Triton 灭活方法，这种方法符合生物安全预期。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Tuberculosis 医学-呼吸系统

CiteScore

4.60

自引率

3.10%

发文量

审稿时长

49 days

期刊介绍： Tuberculosis is a speciality journal focusing on basic experimental research on tuberculosis, notably on bacteriological, immunological and pathogenesis aspects of the disease. The journal publishes original research and reviews on the host response and immunology of tuberculosis and the molecular biology, genetics and physiology of the organism, however discourages submissions with a meta-analytical focus (for example, articles based on searches of published articles in public electronic databases, especially where there is lack of evidence of the personal involvement of authors in the generation of such material). We do not publish Clinical Case-Studies. Areas on which submissions are welcomed include: -Clinical TrialsDiagnostics- Antimicrobial resistance- Immunology- Leprosy- Microbiology, including microbial physiology- Molecular epidemiology- Non-tuberculous Mycobacteria- Pathogenesis- Pathology- Vaccine development. This Journal does not accept case-reports. The resurgence of interest in tuberculosis has accelerated the pace of relevant research and Tuberculosis has grown with it, as the only journal dedicated to experimental biomedical research in tuberculosis.