1Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia.
Jožef Stefan International Postgraduate School, Ljubljana, Slovenia.
mSystems. 2024 Sep 17;9(9):e0073524. doi: 10.1128/msystems.00735-24. Epub 2024 Aug 16.
Targeted high-throughput sequencing (HTS) has revolutionized the way we look at bacterial communities. It can be used for the species-specific detection of bacteria as well as for the determination of the microbiome and resistome and can be applied to samples from almost any environment. However, the results of targeted HTS can be influenced by many factors, which poses a major challenge for its use in clinical diagnostics. In this study, we investigated the impact of the DNA extraction method on the determination of the bacterial microbiome and resistome by targeted HTS using principles from metrology and diagnostics such as repeatability and analytical sensitivity. Sputum samples spiked with , , and at three different concentrations (10-10 cells/mL) were used. DNA was extracted from each sample on 2 separate days in three replicates each using three different extraction methods based on cetrimonium bromide, magnetic beads, and silica membranes. All three spiked bacteria were detected in sputum, and the DNA extraction method had no significant effect on detection. However, the DNA extraction method had significant effects on the composition of the microbiome and the resistome. The sequencing results were repeatable in the majority of cases. The silica membrane-based DNA extraction kit provided the most repeatable results and the highest diversity of the microbiome and resistome. Targeted HTS has been shown to be a reliable tool for determining the microbiome and resistome; however, the method of DNA extraction should be carefully selected to minimize its impact on the results.
High-throughput sequencing (HTS) is one of the crucial new technologies that gives us insights into previously hidden parts of microbial communities. The DNA extraction method is an important step that can have a major impact on the results, and understanding this impact is of paramount importance for their reliable interpretation. Our results are of great value for the interpretation of sputum microbiome and resistome results obtained by targeted HTS. Our findings allow for a more rational design of future microbiome studies, which would lead to higher repeatability of results and easier comparison between different laboratories. This could also facilitate the introduction of targeted HTS in clinical microbiology for reliable identification of pathogenic bacteria and testing for antimicrobial resistance (AMR). As AMR is a major threat to public health, the improved methods for determining AMR would bring great benefits to both the healthcare system and society as a whole.
靶向高通量测序（HTS）彻底改变了我们观察细菌群落的方式。它可用于细菌的种特异性检测，以及微生物组和抗药组的测定，并且可应用于几乎任何环境的样本。然而，靶向 HTS 的结果会受到许多因素的影响，这对其在临床诊断中的应用构成了重大挑战。在这项研究中，我们使用计量学和诊断学的原理（如重复性和分析灵敏度），研究了 DNA 提取方法对靶向 HTS 测定细菌微生物组和抗药组的影响。使用三种不同的提取方法，从三个不同的提取方法中，每种方法重复三次，在两天内从三个浓度（10-10 个细胞/mL）的 、 、 和 痰样中提取 DNA。所有三种加标细菌均在痰中检测到，并且 DNA 提取方法对检测无显著影响。然而，DNA 提取方法对抗药组和微生物组的组成有显著影响。在大多数情况下，测序结果具有重复性。基于硅胶膜的 DNA 提取试剂盒提供了最具重复性的结果和微生物组和抗药组的最高多样性。靶向 HTS 已被证明是一种可靠的工具，可用于确定微生物组和抗药组；然而，应仔细选择 DNA 提取方法，以最大程度地减少其对结果的影响。
高通量测序（HTS）是使我们能够深入了解微生物群落中以前隐藏部分的关键新技术之一。DNA 提取方法是一个重要步骤，它会对结果产生重大影响，因此，正确理解其影响对于可靠地解释结果至关重要。我们的研究结果对于通过靶向 HTS 获得的痰微生物组和抗药组结果的解释具有重要价值。我们的研究结果使人们能够更合理地设计未来的微生物组研究，从而提高结果的重复性，并更容易在不同实验室之间进行比较。这还有助于将靶向 HTS 引入临床微生物学，以可靠地鉴定病原菌并进行抗菌药物耐药性（AMR）检测。由于 AMR 是对公共卫生的重大威胁，因此，确定 AMR 的改进方法将为医疗保健系统和整个社会带来巨大的利益。
