Digital Enrichment of Hyperthermophilic Archaeal Viruses from a Natural Hot Spring Reveals Local Diversification in Response to Host CRISPR-immunity

Despite recent advances in metagenomics, recovering the strain-level diversity of viral communities remains a challenging and laborious process. Metagenomic sequencing approaches are limited by incomplete genome assembly and low coverage for all but the most abundant viral species in a sample. Here, we extend droplet microfluidic cultivation techniques facilitate targeted enrichment of the archaeon Sulfolobus (now Saccharolobus) islandicus and its associated lytic virus S. islandicus rod-shaped virus (SIRV) to enhance viral metagenomics. By compartmentalizing the natural diversity of a Yellowstone hot spring sample into single virus droplets, we eliminate inter-strain competition and preserve population-level diversity during enrichment of the focal viral species. Sequence analysis of enriched metagenomes confirmed that selective enrichment recovered mostly SIRVs, while drop-compartmentalization outperformed a traditional liquid culture to recover the deep local diversity of a lytic viral population from a natural hot spring. Analysis of SIRV variants revealed selective forces shaping the viral population, including local adaptation in response to host CRISPR-immune selection for viral escape mutations.