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

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 (TM). 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 (TM). 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 (TM) 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.

Automated summary

This study demonstrates that full-length 16S rRNA gene sequencing using the MinION(TM) long-read sequencer provides better resolution for bacterial classification, especially in complex gut microbiota.