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

BMC Microbiology

article

Matsuo, Yoshiyuki¹  Naito, Yuji²  Okada, Hidetaka³  Bono, Hidemasa⁴  Nakagawa, So⁶  Hirota, Kiichi¹  Komiya, Shinnosuke⁷  Yasumizu, Yoshiaki⁸  Yasuoka, Yuki⁹  Mizushima, Katsura²  Takagi, Tomohisa²  Kryukov, Kirill⁶  Fukuda, Aisaku^1,11  Morimoto, Yoshiharu⁷ 

[1] Department of Human Stress Response Science, Institute of Biomedical Science, Kansai Medical University;Kyoto Prefectural University of Medicine;Kansai Medical University Graduate School of Medicine;Database Center for Life Science (DBCLS), Research Organization of Information and Systems;Program of Biomedical Science, Graduate School of Integrated Sciences for Life, Hiroshima University;Department of Molecular Life Science, Tokai University School of Medicine;HORAC Grand Front Osaka Clinic;Department of Experimental Immunology, Immunology Frontier Research Center, Osaka University;Faculty of Medicine, Osaka University;Department of Genomics and Evolutionary Biology, National Institute of Genetics;IVF Osaka Clinic

关键词: 16S rRNA;    Gut microbiota;    MinION™;    Nanopore sequencing;   

DOI  :  10.1186/s12866-021-02094-5

学科分类：放射科、核医学、医学影像

来源: BioMed Central

【 摘 要 】

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 MinION™. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. We 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. Our 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.

【 授权许可】

CC BY|CC0