Organelle 16S rRNA amplicon sequencing enables profiling of active gut microbiota in murine model.

High-throughput sequencing of ribosomal RNA (rRNA) amplicons has served as a cornerstone in microbiome studies. Despite crucial implication of organelle 16S rRNA measurements to host gut microbial activities, genomic DNA (gDNA) was overwhelmingly targeted for amplicon sequencings. Although gDNA could be a reliable resource for gene existing validation, little information is revealed in regard to the activity of microorganisms owing to the limited changes gDNA undertaken in inactive, dormant, and dead bacteria. We applied both rRNA- and gDNA-derived sequencings on mouse cecal contents. Respective experimental designs were verified to be suitable for nucleic acid (NA) purification. Via benchmarking, mainstream 16S rRNA hypervariable region targets and reference databases were proven adequate for respective amplicon sequencing study. In phylogenetic studies, significant microbial composition differences were observed between two methods. Desulfovibrio spp. (an important group of anaerobic gut microorganisms that has caused analytical difficulties), Pediococcus spp., and Proteobacteria were drastically lower as represented by gDNA-derived compositions, while microbes like Firmicutes were higher as represented by gDNA-derived microbiome compositions. Also, using PICRUSt2 as an example, we illustrated that rRNA-derived sequencing might be more suitable for microbiome function predictions since pathways like sugar metabolism were lower as represented by rRNA-derived results. The findings of this study demonstrated that rRNA-derived amplicon sequencing could improve identification capability of specific gut microorganisms and might be more suitable for in silico microbiome function predictions. Therefore, rRNA-derived amplicon sequencings, preferably coupled with gDNA-derived ones, could be used as a capable tool to unveil active microbial components in host gut. KEY POINTS: • Conventional pipelines were adequate for the respective amplicon sequencing study • Groups, such as Desulfovibrio spp., were differently represented by two methods • Comparative amplicon sequencings could be useful in host active microbiota studies.