NaMeco - Nanopore full-length 16S rRNA gene reads clustering and annotation

Abstract

Background: Nanopore sequencing is currently one of the leading third-generation sequencing technologies on the market and is gaining popularity among researchers. Due to its long-read capabilities, full-length 16S rRNA gene metabarcoding using Oxford Nanopore Technologies (ONT) offers great potential for metataxonomic studies. However, the relatively high error rate poses a significant challenge for bioinformatic processing, often limiting taxonomy resolution to the genus level despite the longer read length. Results: This study presents NaMeco, a novel tool specifically developed to efficiently process long 16S rRNA gene reads sequenced using Oxford Nanopore Technologies, requiring minimal user input. Our tool performs read quality control, primer-specific extraction of sequences and their clustering, followed by taxonomic annotation with percent identity thresholds that minimize the amount of false-positive annotations. It produces several outputs: a table of cluster counts, taxonomic annotations of clusters, their representative sequences in fasta format and taxa counts at each taxonomy rank. Output files are compatible with the Qiime2 pipeline and can be imported into the required format for downstream analyses. Conclusions: NaMeco, in combination with the full SSU GTDB database, outperforms existing tools such as NanoCLUST and EPI2ME, while delivering taxonomy accuracy and detection rates comparable to Emu.