Large-scale single-cell long-read genomics enables high-resolution microbiome profiling

Microbial communities are extraordinarily diverse and play crucial roles in health and disease, yet current methods lack the resolution and scalability needed to dissect their genomic and ecological complexity at the single-cell level. Here, we present CAP-seq, a high-throughput single-microbe genomics platform that combines hydrogel-based semi-permeable encapsulation with moderate microfluidics to recover thousands of single-amplified genomes (SAGs) with long reads and high completeness at moderate sequencing depth. We benchmarked CAP-seq using defined microbial communities, demonstrating strain-level resolution, accurate detection of rare taxa, and genome recovery exceeding 50% at ~10× coverage. Applying CAP-seq to pediatric Clostridioides difficile infection microbiomes, we generated a high-resolution single-cell atlas comprising tens of thousands of SAGs across hundreds of species. Host-resolved profiling of the cryptic plasmid pBI143 revealed previously hidden low-abundance host associations, four new plasmid versions, and their coexistence within individuals, indicating complex plasmid evolution in situ. Longitudinal analysis during fecal microbiota transplantation and vancomycin treatment uncovered dynamic remodeling of microbial hosts, antimicrobial resistance genes, and plasmid-microbial interactions at single-cell resolution. CAP-seq enables scalable, high-performance single-cell genomics and provides a practical, widely accessible platform for microbiome analysis, paving the way for large-scale exploration of microbial dark matter, host–microbe interactions, and plasmid-microbe dynamics across diverse ecosystems.