NAP: An Open-Source Pipeline for Cross-Domain Microbiome Profiling Using Nanopore Sequencing-Derived Amplicon Data

Background Nanopore sequencing offers a cost-effective and portable platform for amplicon-based microbiome analysis, but is still hindered by limited toolsets and sequencing error profile. While short-read technologies dominate microbial profiling workflows, their portability and flexibility are constrained. There is a need for robust pipelines tailored to Nanopore data that can support cross-kingdom ribosomal RNA profiling. Results We introduce the Nanopore sequencing-based Amplicon Pipeline (NAP; https://github.com/Luke-B-Jones/NAP), an open-source tool optimised for flexible, mixed-domain primer sets (such as 515Y/926R). NAP performs quality filtering, chimera removal, centroid identification, and BLAST-based taxonomic classification with consensus correction. It outputs normalised, bias-corrected tab-separated value files suitable for downstream analysis. Validation against two commercial mock communities showed that NAP achieves genus-level precision of up to 100%, with taxonomic concordance comparable to Illumina-based workflows. Detection sensitivity was consistently reliable above 1% relative abundance. β-diversity measures, including Bray–Curtis and Jaccard indices, fell within expected replicate variation. Taxonomic agreement remained high across a range of read depths and sequencing qualities, with most errors attributable to laboratory-derived artefacts rather than computational limitations. Conclusions NAP delivers robust genus-level performance on par with Illumina workflows, with the potential to achieve species-level resolution using longer amplicons. Its compatibility with portable and cost-effective sequencing makes it well suited for accurate long-read microbiome profiling in both laboratory and field environments.