Abstract

The construction of amplified 16S rRNA gene libraries has been a major methodology for bacterial/archaeal environmental community profiling for decades. Over the years, a variety of alternative primer sets targeting different portions of the rRNA gene have been used. Gradually a widespread collaborative effort, supported by the Earth Microbiome Project (EMP) and other community initiatives, has settled on primers targeting the V4 region (515F/806R) of the 16S rRNA gene, which amplifies both Archaea and Bacteria. Understudied volcanic cave ecosystems possess a high proportion of unclassified bacterial and archaeal lineages, highlighting the selection of optimal primers for community analysis in this environment. Therefore, we investigated the coverage of EMP and alternative archaea-specific (519F/1017R) and bacteria-specific (27F/515R) primers, using microbial and mineral deposits from two volcanic caves. In silico and sequencing analyses showed that the two bacterial primers captured similar amounts and types of Bacteria, while significant differences in the type and abundance of taxa detected between the EMP primers and archaea-specific primers were noted. Our results validate the use of V4 EMP primers for Bacteria in this cave ecosystem, but strongly suggest that Archaea diversity is better captured with the archaea-specific primer in the cave deposits studied.

摘要

几十年来，扩增 16S rRNA 基因库的构建一直是细菌/古菌环境群落分析的主要方法。多年来，已经使用了多种针对 rRNA 基因不同部分的替代引物组。在地球微生物组计划 (EMP) 和其他社区倡议的支持下，一项广泛的合作努力逐渐确定了针对 16S rRNA 基因 V4 区域 (515F/806R) 的引物，该基因可同时扩增古细菌和细菌。未充分研究的火山洞穴生态系统拥有大量未分类的细菌和古菌谱系，突出了在这种环境中选择最佳引物进行群落分析。因此，我们研究了 EMP 和替代古细菌特异性 (519F/1017R) 和细菌特异性 (27F/515R) 引物的覆盖范围，计算机和测序分析表明，两种细菌引物捕获了相似数量和类型的细菌，同时注意到 EMP 引物和古细菌特异性引物之间检测到的类群类型和丰度存在显着差异。我们的结果验证了该洞穴生态系统中细菌的 V4 EMP 引物的使用，但强烈建议使用所研究的洞穴沉积物中的古生菌特异性引物更好地捕获古生菌多样性。