# MitoNGS: an online platform to analyze fish metabarcoding data in high resolution

## TL;DR

MitoNGS is a new online platform that improves the accuracy of identifying fish species from environmental DNA data using advanced analysis methods.

## Contribution

MitoNGS introduces a high-resolution pipeline with expanded reference databases and a 'species complex' strategy to resolve ambiguous taxa in fish metabarcoding.

## Key findings

- MitoNGS improves species-level identification by using comprehensive references and habitat data.
- The platform supports multiple mitochondrial markers and Nanopore sequencing.
- MitoNGS performs well across diverse datasets and sequencing platforms.

## Abstract

Environmental DNA (eDNA) metabarcoding has become a powerful tool for assessing fish biodiversity in aquatic ecosystems. However, accurate species-level identification remains challenging due to incomplete and contaminated reference databases, as well as ambiguous taxa sharing identical barcode sequences. Here, we present MitoNGS, a next-generation platform that succeeds the widely used MiFish pipeline, designed for high-resolution analysis of fish metabarcoding data. MitoNGS addresses these challenges by incorporating more comprehensive references including non-fish species and detailed annotations of heterospecific regions. Additionally, it introduces the “species complex” strategy in conjunction with environmental habitat and geographic occurrence data to resolve ambiguous taxa. Furthermore, MitoNGS expands the functionalities of the legacy MiFish pipeline. It can analyze data from any mitochondrial markers and from Nanopore sequencing platforms. MitoNGS demonstrated excellent performance on our testing datasets from diverse locations, markers, and sequencing platforms. MitoNGS offers a user-friendly, web-based solution for fish detection, biodiversity monitoring, conservation research, and bioresource management. MitoNGS is freely available via https://mitofish.aori.u-tokyo.ac.jp/mito-ngs.