High-coverage, massively parallel sequencing of single-cell genomes with CAP-seq

Abstract

Microorganisms dominate Earth's ecosystems in both abundance and diversity. Studying these organisms relies on their genome information, but obtaining high-quality genomes has long been challenging due to technical limitations in genomic sequencing. Traditional genome sequencing methods are limited by the need to culture isolated strains, excluding unculturable taxa and restricting the study of complex communities. Metagenomics bypasses this but lacks single-cell resolution and often misses rare, critical species. Droplet-based single-cell genomics offers high-throughput genome library preparation but faces challenges like requiring complex microfluidic setups and low genome coverage. To address these challenges, we introduce CAP-seq, a high-throughput single-cell genomic sequencing method with markedly improved genome coverage and ease of use. CAP-seq employs semi-permeable compartments that allow reagent exchange while retaining large DNA fragments, enabling efficient genome processing. This innovation results in higher-quality single amplified genomes (SAGs) and significantly improves resolution. In validation tests with simple and complex microbial communities, CAP-seq yielded high-quality SAGs with over 50% genome coverage, capturing rare taxa more accurately and providing detailed insights into strain-level variation. CAP-seq thus offers a scalable, high-resolution solution for microbial genomic analysis, overcoming the limitations of droplet-based single-cell genomics and enhancing the study of complex microbial ecosystems.