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

Area of Science:

• Microbiology
• Bioinformatics
• Genomics

Background:

• Nanopore sequencing presents a portable and cost-effective method for microbiome analysis.
• Existing amplicon-based methods face challenges with high error rates and lack of tailored workflows for mixed-domain ribosomal RNA profiling.
• Short-read technologies, while dominant, are limited in portability and flexibility for microbial community analysis.

Purpose of the Study:

• To introduce a robust bioinformatics pipeline specifically designed for cross-domain Nanopore amplicon data.
• To address the limitations of existing workflows in handling mixed-domain ribosomal RNA profiling.
• To provide a flexible and accurate tool for microbiome analysis using Nanopore sequencing.

Main Methods:

• Developed the Nanopore sequencing-based Amplicon Pipeline (NAP), an open-source workflow.
• Integrated dynamic quality filtering, base muting, chimera removal, and BLAST-based taxonomic classification.
• Implemented hierarchical consensus correction, read reassignment, and blank-informed decontamination for domain-aware post-processing.

Main Results:

• NAP demonstrated strong performance at the genus level, with reliable recovery above 1% relative abundance.
• The consensus step significantly reduced false positive rates (82.9% at genus level, 78.8% at species level).
• NAP outperformed QIIME2 and Kraken2/Bracken in preserving expected community structure and reducing unexpected genera.

Conclusions:

• NAP offers a reproducible, flexible, and domain-aware consensus workflow for Nanopore amplicon profiling.
• The pipeline shows strongest support at the genus level and competitive species-level performance for well-resolved taxa.
• NAP enhances the utility of Nanopore sequencing for comprehensive microbiome analysis.