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

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.