Esberg Anders, Fries Niklas, Haworth Simon, Johansson Ingegerd
Department of Odontology, Umeå University, Umeå, Sweden.
Bristol Dental School, Bristol, UK.
NPJ Biofilms Microbiomes. 2024 Dec 18;10(1):149. doi: 10.1038/s41522-024-00634-1.
Molecular characterization of the oral microbiome is a crucial first step in experiments which aim to understand the complex dynamics of the oral microbiome or the interplay with host health and disease. Third-generation Oxford Nanopore Technology (ONT) offers advanced long-read sequencing capabilities, which hold promise for improved molecular characterization by distinguishing closely related microbial species within oral ecosystems in health and disease states. However, the performance of ONT sequencing of oral samples requires validation, and the consistency of this approach across different analytical and sampling conditions is not well understood. This study evaluates various factors that may influence the ONT sequencing outputs of saliva microbiota and compares results with those from Illumina MiSeq’s v3v4 amplicon sequencing. Our analysis includes assessments of various stages in the workflow, including different collection and extraction methods, such as robot-extracted saliva DNA used in population-based biobanks, the effects of limited DNA quantities, different bioinformatics pipelines, and different 16S rRNA gene databases. The results demonstrate that ONT provides superior resolution in identifying oral species and subspecies compared to Illumina MiSeq, though the choice of bioinformatics strategy significantly affects the outcomes. Additionally, we confirm the suitability of biobank saliva DNA for large-scale cohort studies, which facilitates the mapping of oral bacterial phylotypes associated with disease states, including less prevalent conditions. Overall, our findings confirm a markedly improved resolution of oral microbiomes by ONT and offer an evidence base to guide the conduct of experiments using this method.
口腔微生物群的分子特征分析是旨在了解口腔微生物群复杂动态或其与宿主健康和疾病相互作用的实验中的关键第一步。第三代牛津纳米孔技术（ONT）提供了先进的长读长测序能力，有望通过区分健康和疾病状态下口腔生态系统中密切相关的微生物物种来改善分子特征分析。然而，口腔样本的ONT测序性能需要验证，并且这种方法在不同分析和采样条件下的一致性尚未得到很好的理解。本研究评估了可能影响唾液微生物群ONT测序输出的各种因素，并将结果与Illumina MiSeq的v3v4扩增子测序结果进行比较。我们的分析包括对工作流程中各个阶段的评估，包括不同的采集和提取方法，如基于人群的生物样本库中使用机器人提取的唾液DNA、DNA数量有限的影响、不同的生物信息学流程以及不同的16S rRNA基因数据库。结果表明，与Illumina MiSeq相比，ONT在识别口腔物种和亚种方面具有更高的分辨率，尽管生物信息学策略的选择会显著影响结果。此外，我们证实了生物样本库唾液DNA适用于大规模队列研究，这有助于绘制与疾病状态相关的口腔细菌系统发育型图谱，包括不太常见的情况。总体而言，我们的研究结果证实了ONT对口腔微生物群分辨率的显著提高，并为指导使用该方法进行实验提供了证据基础。
