Pipeline for species-resolved full-length16S rRNA amplicon nanopore sequencing analysis of low-complexity bacterial microbiota

Abstract 16S rRNA amplicon sequencing is a fundamental tool for characterizing prokaryotic microbial communities. While short-read 16S rRNA sequencing is a proven standard for examining complex microbiomes, it cannot achieve taxonomic classification beyond genus level. Third-generation sequencing technologies, particularly nanopore sequencing, have allowed for full-length 16S rRNA gene sequencing enabling enhanced taxonomic resolution to species and strain levels. Precise species-to-strain level classification is crucial in investigating low-complexity microbiota. This research presents an efficient pipeline using full-length 16S rRNA amplicon nanopore sequencing, spanning library prep to computational analysis for low-complexity microbiota composition analysis. We applied this pipeline to a defined intestinal bacterial community in gnotobiotic mice to evaluate different methods. Our findings revealed that the proprietary barcoded universal primers 27F-1492R from Oxford Nanopore Technologies(ONT) 16S amplicon sequencing kit did not efficiently amplify the 16S rRNA gene of Bifidobacterium species. Addressing this constraint, we designed degenerate primers and employed ONT’s native barcoding kit for library preparation. We developed a customized wet lab and bioinformatics pipeline for processing and classifying amplicon reads at the species level. Validation of the protocol using a mock community DNA sample with known composition confirmed a reduced analytical bias. Additionally, our method surpassed Illumina short-read V3-V4 amplicon sequencing, achieving accurate species-level classification compared to Illumina’s genus-level accuracy. This pipeline is tailored for analyzing the composition of low-complexity microbiota from natural ecosystems and synthetic/gnotobiotic communities. It is cost- and time-effective and therefore accessible for small-scale studies that would otherwise be hindered by the typically long turnaround times of NGS services. Importance 16S rRNA amplicon sequencing is conventionally used to identify microbes and determine their composition in microbial communities. Deep amplicon sequencing of complex microbiomes is well established using short-read sequencing targeting variable regions of the 16S rRNA gene. Short reads enable the classification of bacteria until the genus level in the taxa hierarchy, whereas long reads provide better chances of identifying bacteria to species and even strain levels. This study introduces a streamlined approach for analyzing simple microbial communities using full-length 16S rRNA amplicon nanopore long read sequencing. This approach surpasses Illumina sequencing in species accuracy, is cost-effective and time-efficient. Tailored for low-complexity microbiota, it facilitates studies in natural or synthetic communities, especially beneficial for smaller-scale projects with limited resources.