Metagenomic Hi‑C Protocols for Viral Genome Binning, Taxonomic Annotation, and Interaction Network Visualization.

Metagenomic Hi-C (metaHi-C) links mobile genetic elements to their cellular hosts directly within complex microbial communities. Once shotgun and Hi-C libraries have been generated, however, the main challenges shift to the bioinformatics required for preprocessing, genome binning, taxonomic annotation, and network-level interpretation. Here, we present metaHi-C protocols that span from raw reads to downstream data analyses. Basic Protocol 1 describes quality control of shotgun and Hi-C reads, metagenomic assembly, Hi-C read mapping, and viral contig identification from assembled contigs. Basic Protocol 2 details the use of ViralCC to recover viral metagenome-assembled genomes (vMAGs) and infer virus-host linkages. Support Protocol 1 introduces NormCC and ImputeCC for normalization of raw Hi-C contacts and host genome binning. Support Protocols 2 and 3 describe taxonomic annotation of host MAGs with GTDB-Tk and viral bins with Virgo, respectively. Support Protocol 4 shows how to integrate these outputs in MetaHiCNet to generate cross-taxa and cross-bin Hi-C interaction networks. Together, these protocols provide a reproducible workflow for reconstructing viral and host genomes, assigning consistent taxonomies, and visualizing metaHi-C-derived virus-host interaction structure across diverse microbiomes. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preprocessing raw metagenomic Hi-C data Basic Protocol 2: Viral genome binning and virus-host interaction inference using ViralCC Support Protocol 1: Host genome binning using ImputeCC Support Protocol 2: Host MAG taxonomic annotation with GTDB‑Tk Support Protocol 3: Viral bin taxonomic annotation with Virgo Support Protocol 4: Visualization of virus-host interaction networks with MetaHiCNet.