Data Sheet 1_Comparison of 16S ribosomal RNA hypervariable regions in microbiome studies of anorexia nervosa.pdf

IntroductionShort read sequencing of the 16S ribosomal RNA (rRNA) gene targeting specific hypervariable regions is widely employed to study the human gut microbiota. In these studies, the selection of particular 16S rRNA hypervariable regions is a crucial step. However, the results of such studies exhibit significant variability depending on the targeted hypervariable region.
MethodsShort read sequencing of the 16S ribosomal RNA (rRNA) gene targeting specific hypervariable regions is widely employed to study the human gut microbiota. In these studies, the selection of particular 16S rRNA hypervariable regions is a crucial step. However, the results of such studies exhibit significant variability depending on the targeted hypervariable region.
ResultsThe dominant genera, such as Bacteroides H, Faecalibacterium and Phocaeicola A 858004 were consistently detected in both hypervariable regions across timepoints. The within-sample longitudinal alpha diversity measures varied between the regions with the Chao1 index values being higher in the V1V2 region. The overall microbiome profiles based on beta diversity also differed between the regions. Bland–Altman analysis revealed a general lack of strong agreement between the two sequencing methods, except for a few taxa such as Faecalibacterium, Ruminococcus, Roseburia, Turicibacter and Anaerotruncus. While some results were similar across both hypervariable regions, most of the findings were sensitive to the chosen region.
ConclusionThis study underscores the importance of primer selection in microbiome studies of AN, as it can influence taxonomic resolution and diversity estimates along with downstream statistical analyses.

引言：靶向特定高变区的16S核糖体RNA（16S rRNA）基因短读长测序技术，被广泛应用于人类肠道菌群的研究。在这类研究中，特定16S rRNA高变区的选择是关键步骤之一。然而，此类研究的结果会因所靶向的高变区不同而呈现显著差异。
方法：靶向特定高变区的16S核糖体RNA（16S rRNA）基因短读长测序技术，被广泛应用于人类肠道菌群研究。此类研究中，特定16S rRNA高变区的选择是关键步骤。不过，研究结果会因靶向高变区的不同而存在显著差异。
结果：在不同时间点的两个高变区中，拟杆菌属（Bacteroides）H、粪杆菌属（Faecalibacterium）以及Phocaeicola A 858004等优势菌属均能被稳定检出。样本内纵向α多样性的测定结果因高变区不同存在差异，其中Chao1指数在V1V2区的数值更高。基于β多样性构建的整体菌群谱在不同高变区间也存在显著差异。Bland-Altman分析显示，除粪杆菌属、瘤胃球菌属（Ruminococcus）、罗斯氏菌属（Roseburia）、图里杆菌属（Turicibacter）以及厌氧棍状菌属（Anaerotruncus）等少数类群外，两种测序方法整体缺乏较强的一致性。尽管部分结果在两个高变区间表现相似，但绝大多数研究结果对所选的高变区具有敏感性。
结论：本研究强调了在AN相关菌群研究中引物选择的重要性，因其可影响分类学分辨率、多样性估计以及后续的统计分析。