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

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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: 16S rRNA; Gut microbiota; MinION™; Nanopore sequencing.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
16S rRNA gene sequence analysis using the MinION™ nanopore sequencer. a Workflow of 16S rRNA gene amplicon sequencing on the MinION™ platform. Sequencing libraries are generated by the four-primer PCR-based strategy, enabling simplified post-PCR adapter attachment. At the initial stage of PCR, the 16S rRNA gene is amplified with the inner primer pairs. The resulting PCR products are targeted for amplification with the outer primers to introduce the barcode and tag sequences at both ends, to which adapter molecules can be attached in a single-step reaction. b, c Taxonomic assignments of a mock community analyzed by MinION™ sequencing. The V1-V9 or V3-V4 region of the 16S rRNA gene was amplified from a pre-characterized mock community sample comprising ten bacterial species and sequenced on the MinION™ platform. Three thousand reads were randomly selected from the processed data set and aligned directly to the reference genome database of 5850 representative bacterial species. The pie charts represent taxonomic profiles at the (b) genus and (c) species levels. Even with the full-length 16S rRNA gene analysis, species-level resolution is not possible for Bacillus and Escherichia. Slices corresponding to misclassified (assigned to bacteria not present in the mock community) or unclassified (not classified at the given level but placed in a higher taxonomic rank) reads are exploded. The relative abundance (%) of each taxon is shown
Fig. 2
Fig. 2
Accurate taxonomic assignment afforded by full-length MinION™ sequencing of the 16S rRNA gene. Classification accuracy compared between full-length (V1-V9) and partial (V3-V4) 16S rRNA gene sequencing data obtained from composition profiling of the ten-species mock community. The donut charts show the proportions of reads correctly assigned to the species constituting the mock community. The percentage of correctly classified reads is shown in the center hole. ND: not determined (species-level resolution is not possible for Escherichia)
Fig. 3
Fig. 3
16S rRNA gene sequence analysis of human gut microbiota. Six human fecal samples (F1-F6) were subjected to full-length 16S rRNA gene amplicon sequencing via MinION™. Numbers of detected species are plotted against numbers of reads used for taxonomic classification
Fig. 4
Fig. 4
Comparison of taxonomic profiles of human gut microbiota between sequencing methodologies. Six fecal samples (F1-F6) were analyzed by sequencing the entire 16S rRNA gene using MinION™ (N_V1-V9). For comparison, the V3-V4 region was sequenced on MinION™ (N_V3-V4) or MiSeq™ platforms (I_V1-V9). Randomly sampled 20,000 reads from each data set were allocated to the reference genome database of 5850 representative bacterial species. A heat map shows the relative genus abundance (%) of classified reads. The 15 most abundant taxa are shown. The Pearson correlation coefficient (r) between sequencing methods was computed. Asterisks indicate significant correlations at P < 0.05
Fig. 5
Fig. 5
Comparison of taxonomic resolution. The percentages of ambiguous reads not assigned to the species level are plotted for six fecal samples analyzed by MinION™ (N_V1-V9 and N_V3-V4) or MiSeq™ (I_V3-V4). Horizontal bars represent mean values. * P < 0.05 (statistically significant)
Fig. 6
Fig. 6
Species composition of Bifidobacterium in six fecal samples. MinION™ V1-V9 sequencing confers species-level resolution for bacterial composition profiling. Results obtained by the three sequencing methods are shown. The legends show the 14 most abundant Bifidobacterium species

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