Short-amplicon 16S rRNA gene sequencing is currently the method of choice for studies investigating microbiomes. However, comparative studies on differences in procedures are scarce. We sequenced human stool samples and mock communities with increasing complexity using a variety of commonly used protocols. Short amplicons targeting different variable regions (V-regions) or ranges thereof (V1-V2, V1-V3, V3-V4, V4, V4-V5, V6-V8, and V7-V9) were investigated for differences in the composition outcome due to primer choices. Next, the influence of clustering (operational taxonomic units [OTUs], zero-radius OTUs [zOTUs], and amplicon sequence variants [ASVs]), different databases (GreenGenes, the Ribosomal Database Project, Silva, the genomic-based 16S rRNA Database, and The All-Species Living Tree), and bioinformatic settings on taxonomic assignment were also investigated. We present a systematic comparison across all typically used V-regions using well-established primers. While it is known that the primer choice has a significant influence on the resulting microbial composition, we show that microbial profiles generated using different primer pairs need independent validation of performance. Further, comparing data sets across V-regions using different databases might be misleading due to differences in nomenclature (e.g., Enterorhabdus versus Adlercreutzia) and varying precisions in classification down to genus level. Overall, specific but important taxa are not picked up by certain primer pairs (e.g., Bacteroidetes is missed using primers 515F-944R) or due to the database used (e.g., Acetatifactor in GreenGenes and the genomic-based 16S rRNA Database). We found that appropriate truncation of amplicons is essential and different truncated-length combinations should be tested for each study. Finally, specific mock communities of sufficient and adequate complexity are highly recommended.

中文翻译：

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引物、管道、参数：16S rRNA 基因测序中的问题

短扩增子 16S rRNA 基因测序是目前研究微生物组的首选方法。然而，关于程序差异的比较研究很少。我们使用各种常用协议对人类粪便样本和越来越复杂的模拟社区进行了测序。研究了靶向不同可变区（V 区）或其范围（V1-V2、V1-V3、V3-V4、V4、V4-V5、V6-V8 和 V7-V9）的短扩增子的组成结果差异由于引物的选择。接下来，聚类（操作分类单位 [OTU]、零半径 OTU [zOTU] 和扩增子序列变体 [ASV]）、不同数据库（GreenGenes、核糖体数据库项目、Silva、基于基因组的 16S rRNA 数据库）的影响，以及全物种生命树），还研究了分类分配的生物信息学设置。我们使用完善的引物对所有通常使用的 V 区进行系统比较。虽然众所周知，引物的选择对产生的微生物组成有重大影响，但我们表明，使用不同引物对生成的微生物特征需要独立的性能验证。此外，由于命名法的差异（例如，我们表明使用不同引物对生成的微生物图谱需要独立的性能验证。此外，由于命名法的差异（例如，我们表明使用不同引物对生成的微生物图谱需要独立的性能验证。此外，由于命名法的差异（例如，Enterorhabdus与Adlercreutzia ) 以及不同的分类精度到属级。总体而言，特定的但重要的分类群不会被某些引物对（例如，使用引物 515F-944R 错过拟杆菌）或由于使用的数据库（例如，GreenGenes 中的Acetatifactor和基于基因组的 16S rRNA 数据库）。我们发现对扩增子进行适当的截断是必不可少的，每项研究都应测试不同的截短长度组合。最后，强烈推荐具有足够复杂性的特定模拟社区。