Influence of 16S rRNA Reference Databases in Amplicon-Based Environmental Microbiome Research

Abstract Purpose: The reference databases play a pivotal role in amplicon microbiome research but the sequence content and taxonomic information available in common reference databases differ. Studies on mock community and human health microbiome have revealed the problems associated with the choice of reference database on the outcome. Nonetheless, the influence of reference databases in environmental microbiome studies is not explicitly illustrated. Methods: This study analyzed the amplicon (V1V3, V3V4, V4V5 and V6V8) data of 128 soil samples and evaluated the impact of 16S rRNA databases, Genome Taxonomy Database (GTDB), Ribosomal Database Project (RDP), SILVA and Consensus Taxonomy (ConTax), on microbiome inference. Results: The analyses showed that the distribution of observed amplicon sequence variants was significantly different (P-value < 2.647e-12) across four datasets, generated based on different databases for each amplicon region. In addition, the beta diversity was also found to be altered by different databases. Further investigation revealed that the microbiome composition inferred by different databases vary significantly (P-value=0.001), irrespective of amplicon regions. Importantly, the study found that the core-microbiome structure in environmental studies could be altered by the reference databases. Conclusion: In summary, this present study illustrates that the choice of reference database could influence the outcome of environmental microbiome research.