Abstract Bacterial genome structure changes dynamically, and structural variants can change bacterial phenotype; However, obtaining the complete genome and analyzing genome structure of uncultured bacteria has been challenging. We aimed to develop a single-cell amplified genome long-read assembly (scALA) workflow to construct circular single-cell amplified genomes (cSAGs) from long-read single-cell sequencing data of targeted uncultured bacteria. In particular, scALA generated cSAGs from nanopore long-read sequencing data of SAGs by producing contiguous sequences with repeated bias reduction and assembly processes. From 12 human fecal samples, scALA generated 16 cSAGs of three specifically targeted bacterial species, Anaerostipes hadrus, Agathobacter rectalis , and Ruminococcus gnavus. A. hadrus cSAGs exhibited large, ten kbp-long, phage insertions, saccharide metabolic capacity, and frequent genomic recombination with related strains from cohabitant hosts. Noteworthy, cSAGs constructed using this method could expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.

Revealing within-species diversity in uncultured human gut bacteria with single-cell long-read sequencing