RNA- versus DNA-based 16S rRNA V3-4 amplicon sequencing

Background: Various studies in humans and large animals indicate a relationship between the uterine microbiome composition and endometrial receptivity. The analysis of the uterine microbiome remains challenging due to the very low microbial biomass. Studies of other biological systems showed that RNA-based microbiome analysis complements DNA-based results and provides information about active bacteria in a sample. Thus, the aim of this study was to establish a highly sensitive and specific 16S rRNA gene V3-V4 amplicon PCR from equine uterine cytobrush samples and to compare DNA- and RNA-based 16S rRNA microbiome analysis.Results: A 16S rRNA gene V3-V4 amplicon PCR from equine uterine cytobrush samples was established. Unspecific co-amplification of a host-derived mitochondrial 12S rRNA gene fragment was successfully suppressed. The optimized amplicon PCR protocol was able to detect less than 38 bacterial genome copies using a bacterial DNA community standard. For the RNA-based amplicon generation protocol, an at least 10-fold higher sensitivity was estimated. The comparison of using RNA and DNA isolated from the same uterine cytobrush samples as input for 16S V3-V4 amplicon sequencing revealed a much higher number of amplicon sequence variants as well as taxonomic units for the RNA-based approach. This resulted in significant differences in alpha (Simpson, Chao1) and beta diversity between RNA- and DNA-based analysis. Despite these differences, the overall microbiome composition was similar between the paired DNA and RNA samples. Differential abundance analysis revealed significant differences between DNA and RNA samples at all taxonomic levels.