The multi-batch reanalysis approach of jointly reevaluating gene/genome sequences from different works has gained particular relevance in the literature in recent years. The large amount of 16S ribosomal ribonucleic acid (rRNA) gene sequence data stored in public repositories and information in taxonomic databases of the same gene far exceeds that related to complete genomes. This review is intended to guide researchers new to studying microbiota, particularly the oral microbiota, using 16S rRNA gene sequencing and those who want to expand and update their knowledge to optimise their decision-making and improve their research results. First, we describe the advantages and disadvantages of using the 16S rRNA gene as a phylogenetic marker and the latest findings on the impact of primer pair selection on diversity and taxonomic assignment outcomes in oral microbiome studies. Strategies for primer selection based on these results are introduced. Second, we identified the key factors to consider in selecting the sequencing technology and platform. The process and particularities of the main steps for processing 16S rRNA gene-derived data are described in detail to enable researchers to choose the most appropriate bioinformatics pipeline and analysis methods based on the available evidence. We then produce an overview of the different types of advanced analyses, both the most widely used in the literature and the most recent approaches. Several indices, metrics and software for studying microbial communities are included, highlighting their advantages and disadvantages. Considering the principles of clinical metagenomics, we conclude that future research should focus on rigorous analytical approaches, such as developing predictive models to identify microbiome-based biomarkers to classify health and disease states. Finally, we address the batch effect concept and the microbiome-specific methods for accounting for or correcting them.

中文翻译：

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微生物组研究中 16S rRNA 基因测序工作流程的批判性回顾：从引物选择到高级数据分析

近年来，联合重新评估来自不同作品的基因/基因组序列的多批次重新分析方法在文献中获得了特别的相关性。公共存储库中存储的大量16S核糖体核糖核酸（rRNA）基因序列数据和同一基因的分类数据库中的信息远远超过了完整基因组相关的信息。本综述旨在指导刚开始使用 16S rRNA 基因测序研究微生物群（特别是口腔微生物群）的研究人员，以及那些想要扩展和更新知识以优化决策和改进研究结果的人员。首先，我们描述了使用 16S rRNA 基因作为系统发育标记的优点和缺点，以及口腔微生物组研究中引物对选择对多样性和分类分配结果影响的最新发现。介绍了基于这些结果的引物选择策略。其次，我们确定了选择测序技术和平台时需要考虑的关键因素。详细描述了处理16S rRNA基因衍生数据的主要步骤的过程和特殊性，使研究人员能够根据现有证据选择最合适的生物信息学流程和分析方法。然后，我们概述了不同类型的高级分析，包括文献中使用最广泛的方法和最新的方法。其中包括一些用于研究微生物群落的指数、指标和软件，强调了它们的优点和缺点。考虑到临床宏基因组学的原理，我们得出的结论是，未来的研究应侧重于严格的分析方法，例如开发预测模型来识别基于微生物组的生物标志物，以对健康和疾病状态进行分类。最后，我们讨论了批次效应的概念以及用于解释或纠正它们的微生物组特定方法。