Benchmark of 16S rRNA gene amplicon sequencing using Japanese gut microbiome data from the V1–V2 and V3–V4 primer sets

Abstract

Background 16S rRNA gene amplicon sequencing (16S analysis) is widely used to analyze microbiota with next-generation sequencing technologies. Here, we compared fecal 16S analysis data from 192 Japanese volunteers using the modified V1–V2 (V12) and the standard V3–V4 primer (V34) sets to optimize the gut microbiota analysis protocol. Results QIIME1 and QIIME2 analysis revealed a higher number of unclassified representative sequences in the V34 data than in the V12 data. The comparison of bacterial composition demonstrated that at the phylum level, Actinobacteria and Verrucomicrobia were detected at higher levels with V34 than with V12. Among these phyla, we observed higher relative compositions of Bifidobacterium and Akkermansia with V34. To estimate the actual abundance, we performed quantitative real-time polymerase chain reaction (qPCR) assays for Akkermansia and Bifidobacterium. We found that the abundance of Akkermansia as detected by qPCR was close to that in V12 data, but was markedly lower than that in V34 data. The abundance of Bifidobacterium detected by qPCR was higher than that in V12 and V34 data. Conclusions These results indicate that the bacterial composition derived from the V34 region might differ from the actual abundance for specific gut bacteria. We conclude that the use of the modified V12 primer set is more desirable in the 16S analysis of the Japanese gut microbiota.

Choice of 16S ribosomal RNA primers impacts urinary microbiota profiling

“…Many studies have compared the performance of different sets of 16S rRNA primers for microbiota profiling in different environments (10)(11)(12)(13). These studies consistently demonstrated that the choice of the 16S rRNA primer set can significantly influence the analysis of microbiota diversity and composition.…”

“…Although all nine hypervariable regions are taxonomically informative, the amount and quality of information retrieved varies per region according to the studied environment. For instance, Fadeev et al (2021) showed that V4V5 is superior to V3V4 for microbiota profiling of environmental arctic samples (10), and Kameoka et al (2021) found that V1V2 is more precise than V3V4 for gut microbiota profiling of Japanese individuals (11).…”

“…Despite evidence showing that the choice of 16S hypervariable regions in microbiota profiling studies is critical (10)(11)(12)(13), no study has systematically compared the performance of different 16S rRNA hypervariable regions for microbial characterization of urine samples. In this work, we compared the performance of different sets of 16S rRNA primers for urinary microbiota profiling.…”

Ketogenic Effects of Multiple Doses of a Medium Chain Triglycerides Enriched Ketogenic Formula in Healthy Men under the Ketogenic Diet: A Randomized, Double-Blinded, Placebo-Controlled Study

“…DNA extraction from the fecal samples was performed using an automated DNA extraction system (GENEPREP STAR PI-480, Kurabo Industries Ltd., Osaka, Japan) according to the manufacturer’s protocols. The V1–V2 region of the 16S rRNA gene was amplified using a forward primer (16S_27Fmod: TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG AGR GTT TGA TYM TGG CTC AG) and a reverse primer (16S_338R: GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GTG CTG CCT CCC GTA GGA GT) and KAPA HiFi HotStart ReadyMix (Roche, Basel, CHE) according to a previous study [ 13 ]. To sequence 16S amplicons using the Illumina MiSeq platform, dual index adapters were attached using the Nextera XT Index Kit.…”

Metataxonomic investigation of the microbial community in the trachea and oropharynx of healthy controls and diabetic patients using endotracheal tubes

“…In this study, we selected the V3-V4 region in consideration of the longest read length and cost, but there is a limitation in not investigating the difference in results that may occur when other regions are targeted. In Partial 16s rRNA gene sequencing, the difference in results pertaining to the selection of hypervariable regions of the 16S rRNA gene on various clinical samples has been previously reported [44,45]. For respiratory samples, the V1-V2 region was selected for better species-level taxonomic assignment in a study with endotracheal aspirates and oropharyngeal swab [40].…”