Abstract：
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.
摘要：
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。存储在公共存储库中的大量16S核糖体核糖核酸（rRNA）基因序列数据以及同一基因的分类数据库中的信息远远超过与完整基因组相关的信息。这篇综述旨在指导研究微生物群的新研究人员，特别是口腔微生物群，使用16SrRNA基因测序和那些想要扩展和更新知识的人，以优化他们的决策并改善他们的研究结果。首先,我们描述了使用16SrRNA基因作为系统发育标记的优势和劣势,以及在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的影响的最新发现.介绍了基于这些结果的引物选择策略。第二,我们确定了选择测序技术和平台需要考虑的关键因素。详细描述了处理16SrRNA基因衍生数据的主要步骤的过程和特殊性，以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，文献中使用最广泛的方法和最新的方法。几个指数，包括用于研究微生物群落的指标和软件，突出它们的优点和缺点。考虑到临床宏基因组学的原理，我们得出结论，未来的研究应该集中在严格的分析方法上，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,我们讨论了批量效应的概念和微生物组特有的方法来解释或纠正它们。
