<scp>HiMBar</scp> : A High‐Fidelity Metagenomic Barcoding Approach for Transkingdom Species Detection and Interaction Analysis in Aquatic Ecosystems

Aquatic ecosystems host diverse organisms across all six life kingdoms, yet their complex interactions remain poorly understood, primarily due to limitations in transkingdom species detection methods. To address this limitation, we developed HiMBar (https://github.com/Xchenkai2019/HIFI_barcoding), a high-fidelity (HiFi) metagenomic barcoding approach that utilises long, highly accurate reads to extract multiple full-length marker genes (such as rRNA genes, COI, rbcL) directly from environmental DNA sequencing reads. These genes are subsequently clustered into operational taxonomic units (OTUs) for species identification, eliminating the need for PCR amplification or sequence assembly. HiMBar outperforms existing DNA-based methods in accuracy, recall and consistency. Applying HiMBar, we identified a stable interaction network among Cyanobacteria, Planctomycetota, Verrucomicrobiota and Fungi. Further analysis revealed that glucose metabolism plays a key role in maintaining these interactions. Our study offers a powerful tool for transkingdom species monitoring and provides a case study for exploring transkingdom interactions and their molecular mechanisms.