Divergent Bacteriophages from Wastewater Reveal an Open Pan-Genome with No Shared Gene Families

Bacteriophages, the most abundant and genetically diverse biological entities, have much of their diversity unexplored. Wastewater systems with intact microbial com-munities serve as reservoirs for diverse phages. This study recovered complete phage genomes from rural wastewater metagenomes to assess their genetic diversity. Meta-genomic sequences from six wastewater samples were assembled, viral contigs identi-fied using PHASTEST, yielding 17 high-confidence phage genomes. These were anno-tated and compared via pangenome analysis, gene-sharing networks, phylogenetic re-construction, and average amino acid identity. Heaps’ law and UpSet plots quantified pangenome openness and gene family intersections. The 17 phage genomes encoded 30–172 proteins each, sharing no core genes. Of 1,031 gene families, 98.7% were “cloud” and 94% unique, with only 13 “shell” families in >2 phages. Most shared no genes (average Jaccard similarity < 1%), 15 appearing as isolated nodes in networks. Phylogenetic trees exhibited star-like topology, reflecting distinct paths. The pange-nome was open (Heaps’ law α ≈ 0.026), with minimal overlap confirmed by UpSet, and ancestral reconstruction indicated stable genomes with occasional gains. In conclusion, bacteriophages exhibit extreme genomic diversity even in one environment, each ge-nome largely unique, highlighting the immense uncharted phage diversity and sup-porting high diversity in varied habitats.