Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION\u2122 nanopore sequencing confers species-level resolution

Yoshiyuki Matsuo,Hidemasa Bono,Hidetaka Okada,Yuji Naito,Kiichi Hirota,Shinnosuke Komiya,Aisaku Fukuda,So Nakagawa,Tomohisa Takagi,Yuki Yasuoka,Yoshiaki Yasumizu,Yoshiharu Morimoto,Kirill Kryukov,Katsura Mizushima

doi:10.1186/s12866-021-02094-5

Abstract

BackgroundSpecies-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples.ResultsWe modified our existing protocol for full-length 16S rRNA gene amplicon sequencing by MinION™. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S rRNA gene amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION™ long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition.ConclusionsOur present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene.

Highlights

Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment
The 16S ribosomal RNA (rRNA) gene is first amplified by polymerase chain reaction (PCR) with primers annealing to conserved regions and sequenced
Classification of the mock bacterial community The 16S rRNA gene sequence of Bifidobacterium has three base mismatches with the 27F forward primer provided in the commercial sequencing kit (16S Barcoding Kit, SQK-RAB204, Oxford Nanopore Technologies; Additional File 2: Supplementary Fig. S1a), which biases amplification toward underrepresentation of Bifidobacterium species (Additional File 2: Supplementary Fig. S2, Additional File 3: Supplementary Table S1-S3)

Summary

Introduction

Species-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinIONTM. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. Metagenomic sequencing offers numerous advantages over traditional culture-based techniques that have long been the standard test for detecting pathogenic bacteria. This method is useful for characterizing uncultured bacteria and novel pathogens [3]. The sequencing data are subjected to bioinformatic analysis in which the variable regions are used to discriminate between bacterial taxa [6]

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