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Enrichment of gut microbiome strains for cultivation-free genome sequencing using droplet microfluidics.

ABSTRACT: We report a droplet microfluidic method to target and sort individual cells directly from complex microbiome samples and to prepare these cells for bulk whole-genome sequencing without cultivation. We characterize this approach by recovering bacteria spiked into human stool samples at a ratio as low as 1:250 and by successfully enriching endogenous Bacteroides vulgatus to the level required for de novo assembly of high-quality genomes. Although microbiome strains are increasingly demanded for biomedical applications, a vast majority of species and strains are uncultivated and without reference genomes. We address this shortcoming by encapsulating complex microbiome samples directly into microfluidic droplets and amplifying a target-specific genomic fragment using a custom molecular TaqMan probe. We separate those positive droplets by droplet sorting, selectively enriching single target strain cells. Finally, we present a protocol to purify the genomic DNA while specifically removing amplicons and cell debris for high-quality genome sequencing.

SUBMITTER: Pryszlak A

PROVIDER: S-EPMC8787643 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Enrichment of gut microbiome strains for cultivation-free genome sequencing using droplet microfluidics.

Pryszlak Anna A Wenzel Tobias T Seitz Kiley West KW Hildebrand Falk F Kartal Ece E Cosenza Marco Raffaele MR Benes Vladimir V Bork Peer P Merten Christoph A CA

Cell reports methods 20220124 1

We report a droplet microfluidic method to target and sort individual cells directly from complex microbiome samples and to prepare these cells for bulk whole-genome sequencing without cultivation. We characterize this approach by recovering bacteria spiked into human stool samples at a ratio as low as 1:250 and by successfully enriching endogenous <i>Bacteroides vulgatus</i> to the level required for de novo assembly of high-quality genomes. Although microbiome strains are increasingly demanded ...[more]

PMID: 35118437

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Project description:BackgroundDespite the importance of the mucosal interface between microbiota and the host in gut homeostasis, little is known about the mechanisms of bacterial gut colonization, involving foraging for glycans produced by epithelial cells. The slow pace of progress toward understanding the underlying molecular mechanisms is largely due to the lack of efficient discovery tools, especially those targeting the uncultured fraction of the microbiota.ResultsHere, we introduce an ultra-high-throughput metagenomic approach based on droplet microfluidics, to screen fosmid libraries. Thousands of bacterial genomes can be covered in 1 h of work, with less than ten micrograms of substrate. Applied to the screening of the mucosal microbiota for β-N-acetylgalactosaminidase activity, this approach allowed the identification of pathways involved in the degradation of human gangliosides and milk oligosaccharides, the structural homologs of intestinal mucin glycans. These pathways, whose prevalence is associated with inflammatory bowel diseases, could be the result of horizontal gene transfers with Bacteroides species. Such pathways represent novel targets to study the microbiota-host interactions in the context of inflammatory bowel diseases, in which the integrity of the mucosal barrier is impaired.ConclusionBy compartmentalizing experiments inside microfluidic droplets, this method speeds up and miniaturizes by several orders of magnitude the screening process compared to conventional approaches, to capture entire metabolic pathways from metagenomic libraries. The method is compatible with all types of (meta)genomic libraries, and employs a commercially available flow cytometer instead of a custom-made sorting system to detect intracellular or extracellular enzyme activities. This versatile and generic workflow will accelerate experimental exploration campaigns in functional metagenomics and holobiomics studies, to further decipher host-microbiota relationships. Video Abstract.

Dataset Information

Enrichment of gut microbiome strains for cultivation-free genome sequencing using droplet microfluidics.

Publications

Enrichment of gut microbiome strains for cultivation-free genome sequencing using droplet microfluidics.

Similar Datasets

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