Choice of 16S Ribosomal RNA Primers Impacts Male Urinary Microbiota Profiling

Abstract

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 male 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. Systematic comparisons of their performance indicate V1V2 hypervariable regions better assess the taxa commonly present in male urine samples, suggesting V1V2 amplicon sequencing is more suitable for male urinary microbiota profiling. We believe our results will be helpful to guide this crucial methodological choice in future male urinary microbiota studies.

Improved Assembly of Metagenome-Assembled Genomes and Viruses in Tibetan Saline Lake Sediment by HiFi Metagenomic Sequencing

“…We applied the selected method rather than previously applied rRNA-based species community survey technologies for the following reasons: (i) There is no requirement for PCR in HiFi sequencing library construction, while there are at least 2 PCR steps in the NGS sequencing protocol, which introduces community bias caused by high GC contents and complex gene structures ( 48 ). (ii) NGS-based rRNA gene recovery requires multiple types of primers to amplify hypervariable regions, and profiling performance varies in different habitats ( 49 ). Moreover, rRNA reads from metagenomic studies provide a source of sequences that are not subject to PCR primer bias and therefore cover taxa that might be missed with existing primer sets ( 50 ).…”

Urinary microbiota is associated to clinicopathological features in benign prostatic hyperplasia

“…Of note, detectability was considerably higher in our cohort (76% vs. 27%), likely reflecting the optimized protocol we adopted. 19 Chronic prostatic inflammation has been associated with BPH pathogenesis and clinical progression. [12][13][14] Although the mechanisms inducing inflammation are not yet completely understood, recent studies suggest a role of the urinary microbiota in this process.…”

“…16S/ITS Region Panel kit (Qiagen). We chose to amplify and sequence the 16S gene hypervariable regions V1V2, as recommended for male urinary samples 19 . Libraries with a final concentration <0.4 nM were deemed to have undetectable microbiota and were not sequenced.…”

“…We chose to amplify and sequence the 16S gene hypervariable regions V1V2, as recommended for male urinary samples. 19 Libraries with a final concentration <0.4 nM were deemed to have undetectable microbiota and were not sequenced. Libraries with detectable microbes were sequenced at a final concentration of 10 pM in the Illumina MiSeq System using the MiSeq Reagent Kit v3.…”

“…23 Finally, contaminant ASVs were identified in negative controls and removed as described previously. 19…”