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

Masato Kogawa; Yohei Nishikawa; Tatsuya Saeki; Takuya Yoda; Koji Arikawa; Haruko Takeyama; Masahito Hosokawa

doi:10.3389/fmicb.2023.1133917

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

Front Microbiol. 2023 Feb 24:14:1133917. doi: 10.3389/fmicb.2023.1133917. eCollection 2023.

Authors

Masato Kogawa¹, Yohei Nishikawa^{1

2}, Tatsuya Saeki³, Takuya Yoda³, Koji Arikawa³, Haruko Takeyama^{1

2

4

5}, Masahito Hosokawa^{1

2

3

4

5}

Affiliations

¹ Research Organization for Nano and Life Innovation, Waseda University, Tokyo, Japan.
² Computational Bio Big-Data Open Innovation Laboratory, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan.
³ bitBiome, Inc., Tokyo, Japan.
⁴ Department of Life Science and Medical Bioscience, Waseda University, Tokyo, Japan.
⁵ Institute for Advanced Research of Biosystem Dynamics, Waseda Research Institute for Science and Engineering, Tokyo, Japan.

Abstract

Obtaining complete and accurate bacterial genomes is vital for studying the characteristics of uncultured bacteria. Single-cell genomics is a promising approach for the culture-independent recovery of bacterial genomes from individual cells. However, single-amplified genomes (SAGs) often have fragmented and incomplete sequences due to chimeric and biased sequences introduced during the genome amplification process. To address this, we developed a single-cell amplified genome long-read assembly (scALA) workflow to construct complete circular SAGs (cSAGs) from long-read single-cell sequencing data of uncultured bacteria. We used the SAG-gel platform, which is both cost-effective and high-throughput, to obtain hundreds of short-read and long-read sequencing data for specific bacterial strains. The scALA workflow generated cSAGs by repeated in silico processing for sequence bias reduction and contig assembly. From 12 human fecal samples, including two cohabitant groups, scALA generated 16 cSAGs of three specifically targeted bacterial species: Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. We discovered strain-specific structural variations shared among cohabiting hosts, while all cSAGs of the same species showed high homology in aligned genomic regions. A. hadrus cSAGs exhibited 10 kbp-long phage insertions, various saccharide metabolic capabilities, and different CRISPR-Cas systems in each strain. The sequence similarity of A. hadrus genomes did not necessarily correspond with orthologous functional genes, while host geographical regionality seemed to be highly related to gene possession. scALA allowed us to obtain closed circular genomes of specifically targeted bacteria from human microbiota samples, leading to an understanding of within-species diversities, including structural variations and linking mobile genetic elements, such as phages, to hosts. These analyses provide insight into microbial evolution, the adaptation of the community to environmental changes, and interactions with hosts. cSAGs constructed using this method can expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.

Keywords: genome; gut; long-read sequencing; microbiome; single-cell genomics.