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Abstract Background The increase in metagenome-assembled genomes (MAGs) has advanced our understanding of the functional characterization and taxonomic assignment within the human microbiome. However, MAGs, as population consensus genomes, often aggregate heterogeneity among species and strains, thereby obfuscating the precise relationships between microbial hosts and mobile genetic elements (MGEs). In contrast, single amplified genomes (SAGs) derived via single-cell genome sequencing can capture individual genomic content, including MGEs. Results We introduce the first substantial SAG dataset (bbsag20) from the human oral and gut microbiome, comprising 17,202 SAGs above medium-quality without co-assembly. This collection unveils a diversity of bacterial lineages across 312 oral and 647 gut species, demonstrating different taxonomic compositions from MAGs. Moreover, the SAGs showed cellular-level evidence of the translocation of oral bacteria to the gut. We also identified broad-host-range MGEs harboring antibiotic resistance genes (ARGs), which were not detected in the MAGs. Conclusions The difference in taxonomic composition between SAGs and MAGs indicates that combining both methods would be effective in expanding the genome catalog. By connecting mobilomes and resistomes in individual samples, SAGs could meticulously chart a dynamic network of ARGs on MGEs, pinpointing potential ARG reservoirs and their spreading patterns in the microbial community. Video Abstract

Abstract Background Obtaining high-quality (HQ) reference genomes from microbial communities is crucial for understanding the phylogeny and function of uncultured microbes in complex microbial ecosystems. Despite improvements in bioinformatic approaches to generate curated metagenome-assembled genomes (MAGs), existing metagenome binners obtain population consensus genomes but they are nowhere comparable to genomes sequenced from isolates in terms of strain level resolution. Here, we present a framework for the integration of single-cell genomics and metagenomics, referred to as single-cell (sc) metagenomics, to reconstruct strain-resolved genomes from microbial communities at once. Results Our sc-metagenomics integration framework, termed SMAGLinker, uses single-cell amplified genomes (SAGs) generated using microfluidic technology as binning guides and integrates them with metagenome-assembled genomes (MAGs) to recover improved draft genomes. We compared sc-metagenomics with the metagenomics-alone approach using conventional metagenome binners. The sc-metagenomics approach showed precise contig binning and higher recovery rates (>97%) of rRNA and plasmids than conventional metagenomics in genome reconstruction from the cell mock community. In human microbiota samples, sc-metagenomics recovered the largest number of genomes with a total of 103 gut microbial genomes (21 HQ, with 65 showing >90% completeness) and 45 skin microbial genomes (10 HQ, with 40 showing >90% completeness), respectively. Conventional metagenomics recovered one Staphylococcus hominis genome, whereas sc-metagenomics recovered two S. hominis genomes from identical skin microbiota sample. Single-cell sequencing revealed that these S. hominis genomes were derived from two distinct strains harboring specifically different plasmids. We found that all conventional S. hominis MAGs had a substantial lack or excess of genome sequences and contamination from other Staphylococcus species (S. epidermidis). Conclusions SMAGLinker enabled us to obtain strain-resolved genomes in the mock community and human microbiota samples by assigning metagenomic sequences correctly and covering both highly conserved genes such as rRNA genes and unique extrachromosomal elements, including plasmids. SMAGLinker will provide HQ genomes that are difficult to obtain using metagenomics alone and will facilitate the understanding of microbial ecosystems by elucidating detailed metabolic pathways and horizontal gene transfer networks. SMAGLinker is available at https://github.com/kojiari/smaglinker . Video abstract