In:
Science, American Association for the Advancement of Science (AAAS), Vol. 376, No. 6597 ( 2022-06-03)
Abstract:
The human gut microbiome is a complex ecosystem specific to each individual that comprises hundreds of microbial species. Different strains of the same species can impact health disparately in important ways, such as through antibiotic resistance and host-microbiome interactions. Consequently, consideration of microbes only at the species level without identifying their strains obscures important distinctions. The strain-level genomic structure of the gut microbiome has yet to be elucidated fully, even within a single person. Shotgun metagenomics broadly surveys the genomic content of microbial communities but in general cannot capture strain-level variations. Conversely, culture-based approaches and titer plate-based single-cell sequencing can yield strain-resolved genomes, but access only a limited number of microbial strains. RATIONALE We develop and validate Microbe-seq—a high-throughput single-cell sequencing method with strain resolution—and apply it to the human gut microbiome. Using an integrated microfluidic workflow, we encapsulate tens of thousands of microbes individually into droplets. Within each droplet, we lyse the microbe, perform whole-genome amplification, and tag the DNA with droplet-specific barcodes; we then pool the DNA from all droplets and sequence. In mammalian systems—the focus of most single-cell studies—high-quality reference genomes are available for the small number of species under investigation; by contrast, in complex communities of 100 or more microbial species—such as the human gut microbiome—reference genomes are a priori unknown. Therefore, we develop a generalizable computational framework that combines sequencing reads from multiple microbes of the same species to generate a comprehensive list of reference genomes. By comparing individual microbes from the same species, we identify whether multiple strains coexist and coassemble their strain-resolved genomes. The resulting collection of high-quality strain-resolved genomes from a broad range of microbial taxa enables the ability to probe, in unprecedented detail, the genomic structure of the microbial community. RESULTS We apply Microbe-seq to seven gut microbiome samples collected from one human subject and acquire 21,914 single-amplified genomes (SAGs), which we coassemble into 76 species-level genomes, many from species that are difficult to culture. Ten of these species include multiple strains whose genomes we coassemble. We use these strain-resolved genomes to reconstruct the horizontal gene transfer (HGT) network of this microbiome; we find frequent exchange among Bacteroidetes species related to a mobile element carrying a Type-VI secretion system, which mediates inter-strain competition. Our droplet-based encapsulation also provides the opportunity to probe physical associations between individual microbes and colocalized bacteriophages. We find a significant host-phage association between crAssphage, the most abundant bacteriophage known in the human gut microbiome, and one particular strain of Bacteroides vulgatus . CONCLUSION We use Microbe-seq, combining microfluidic-droplet operation with tailored bioinformatic analysis, to achieve a strain-resolved survey of the genomic structure of a single person’s gut microbiome. Our methodology is general and immediately applicable to other complex microbial communities, such as the microbiomes in the soil and ocean. Applying our method to a broader human population and integrating Microbe-seq with other techniques, including functional screening, sorting, and long-read sequencing, could significantly enhance the understanding of the gut microbiome and its interaction with human health. Microbe-seq overview. Cells encapsulated individually at high throughput into droplets are lysed and resulting DNA amplified and barcoded. Pooled DNA sequencing yields single amplified genomes, which are clustered and coassembled into reference genomes of ~100 species. For multistrain species, assigning SAGs to constituent strains through SNPs enables coassembly of strain-resolved genomes, used to elucidate the HGT network and host-phage associations.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.abm1483
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2022
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
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