Choice of 16S ribosomal RNA primers impacts urinary microbiota profiling

Accessibility to next-generation sequencing (NGS) technologies has enabled the profiling of microbial communities living in distinct habitats. 16S ribosomal RNA (rRNA) gene sequencing is widely used for microbiota profiling with NGS technologies. Since most used NGS platforms generate short reads, sequencing the full-length 16S rRNA gene is impractical. Therefore, choosing which 16S rRNA hypervariable region to sequence is critical in microbiota profiling studies. All nine 16S rRNA hypervariable regions are taxonomically informative, but due to variability in profiling performance for specific clades, choosing the ideal 16S rRNA hypervariable region will depend on the bacterial composition of the habitat under study. Recently, NGS allowed the identification of microbes in the urinary tract, and urinary microbiota has become an active research area. However, there is no current study evaluating the performance of different 16S rRNA hypervariable regions for urinary microbiota profiling. We collected urine samples from male volunteers and profiled their urinary microbiota by sequencing a panel of six amplicons encompassing all nine 16S rRNA hypervariable regions. After systematically comparing their performance, we show that V1V2 hypervariable regions better assess the taxa commonly present in urine samples and V1V2 amplicon sequencing is more suitable for urinary microbiota profiling. We believe our results will be helpful to guide this crucial methodological choice in future urinary microbiota studies.