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

Matsuo, Y.; Komiya, S.; Yasumizu, Y.; Yasuoka, Y.; Mizushima, K.; Takagi, T.; Kryukov, K.; Fukuda, A.; Morimoto, Y.; Naito, Y.; Okada, H.; Bono, H.; Nakagawa, S.; Hirota, K.

doi:10.1101/2020.05.06.078147

bioRxiv · 10.1101/2020.05.06.078147

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

Matsuo, Y.·Komiya, S.·Yasumizu, Y.·Yasuoka, Y.·Mizushima, K.·Takagi, T.·Kryukov, K.·Fukuda, A.·Morimoto, Y.·Naito, Y.·Okada, H.·Bono, H.·Nakagawa, S.·Hirota, K.

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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 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 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, which provided the requisite species-level resolution and accuracy in clinical settings.

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Matsuo, Y., Komiya, S., Yasumizu, Y., Yasuoka, Y., Mizushima, K., Takagi, T., Kryukov, K., Fukuda, A., Morimoto, Y., Naito, Y., Okada, H., Bono, H., Nakagawa, S., Hirota, K.. 2020-05-08. Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution. https://doi.org/10.1101/2020.05.06.078147

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