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research article

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

Pryszlak, Anna
•
Wenzel, Tobias
•
Seitz, Kiley West
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January 24, 2022
Cell Reports Methods

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.

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1-s2.0-S266723752100206X-main.pdf

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http://purl.org/coar/version/c_970fb48d4fbd8a85

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openaccess

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CC BY-NC-ND

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3.45 MB

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