Abstract Introduction Due to recent improvements, Nanopore sequencing has become a promising method for experiments relying on amplicon sequencing. We describe a flexible workflow to generate and annotate high-quality, full-length 16S rDNA amplicons. We evaluated it for two applications, namely, i) identification of bacterial isolates and ii) species-level profiling of microbial communities. Methods Bacterial isolate identification by sequencing was tested on 47 isolates and compared to MALDI-TOF MS. 97 isolates were additionally sequenced to assess the resolution of phylogenetic classification. Species-level community profiling was tested with two full-length 16S primer pairs (A and B) with custom barcodes and compared to results obtained with Illumina sequencing using 27 stool samples. Finally, a Nextflow pipeline was developed to produce high-quality reads and taxonomically annotate them. Results We found high agreement between our workflow and MALDI-TOF data for isolate identification (PPV = 0.90, Cramér’s V = 0.857 and, Theil’s U = 0.316). For species-level community profiling, we found strong correlations (r s &gt; 0.6) of alpha diversity indices between the two primer sets and Illumina sequencing. At the community level, we found significant but small differences when comparing sequencing techniques. Finally, we found moderate to strong correlation when comparing relative abundances of individual species (average r s = 0.6 and 0.533, for primers A and B). Discussion The proposed workflow enabled accurate identification of single bacterial isolates, making it a worthwhile alternative to MALDI-TOF. While shortcomings have been identified, it enabled reliable identification of prominent features in microbial communities at a fraction of the cost of Illumina sequencing. Importance A quick, robust, simple, and cost-effective method to identify bacterial isolates and communities in each sample is indispensable in the fields of microbiology and infection biology. Recent technological advances in Oxford Nanopore Technologies sequencing make this technique an attractive option considering the adaptability, portability, and cost-effectiveness of the platform. Here, we validated a flexible workflow to identify bacterial isolates and characterize bacterial communities using the Oxford Nanopore Technologies sequencing platform combined with the most recent v14 chemistry kits. For bacterial isolates, we compared our nanopore-based approach to MALDI-TOF MS-based identification. For species-level profiling of complex bacterial communities we compared our nanopore-based approach to Illumina shotgun sequencing. For reproducibility purposes, we wrapped the code used to process the sequencing data into a ready-to-use and self-contained Nextflow pipeline.

A novel barcoded nanopore sequencing workflow of high-quality, full-length bacterial 16S amplicons for taxonomic annotation of bacterial isolates and complex microbial communities