NAP: an open-source pipeline for cross-domain microbiome profiling using Nanopore sequencing-derived amplicon data

BackgroundNanopore sequencing offers a cost-effective and portable platform for microbiome analysis, but amplicon-based approaches remain limited by higher sequencing error rates and a lack of workflows tailored to mixed domain ribosomal RNA profiling. While short-read technologies dominate microbial community analysis, their portability and flexibility are constrained. There is therefore a need for robust pipelines designed specifically for cross-domain Nanopore amplicon data. ResultsWe introduce the Nanopore sequencing-based Amplicon Pipeline (NAP; https://github.com/Luke-B-Jones/NAP), an open-source workflow optimised for flexible mixed domain primer sets such as 515Y/926R. NAP performs adaptive quality filtering, chimera removal, centroid generation, BLAST-based taxonomic classification, hierarchical consensus correction, and domain-aware post-processing, outputting decontaminated abundance tables suitable for downstream analysis. Initial validation against two complementary commercial mock communities showed that NAP achieved strong genus-level performance across both low complexity logarithmic and more compositionally complex gut mock communities. Detection was most reliable above ca. 1% relative abundance, and replicate outputs showed strong agreement with expected composition under Bray-Curtis, Jaccard, agreement-plot, and Bland-Altman analyses. Benchmarking of NAPs internal filtering modes showed that the default adaptive setting provided the most robust balance of read quality, retained depth, and downstream taxonomic fidelity across heterogeneous inputs. Direct comparison against QIIME2 and Kraken2/Bracken further showed that NAP most accurately preserved expected community structure, with markedly fewer false positive assignments at genus level and substantially stronger species-level behaviour under the tested conditions. Species-level assignments were informative for some taxa, but remained less robust than genus-level outputs with the default V4-V5 amplicon. ConclusionsNAP provides a robust and flexible workflow for cross-domain Nanopore amplicon profiling, with strongest performance at genus level and competitive species-level behaviour for well resolved taxa. Although analysis of field-derived data was not assessed here, NAP compatibility with portable Nanopore sequencing supports accurate mixed domain microbiome profiling under the tested conditions.