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arXiv · 2106.01455

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

Anna Pryszlak·Tobias Wenzel·Kiley West Seitz·Falk Hildebrand·Ece Kartal·Marco Raffaele Cosenza·Vladimir Benes·Peer Bork·Christoph Merten
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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. While microbiome strains are increasingly demanded for biomedical applications, the 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 amplify 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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Anna Pryszlak, Tobias Wenzel, Kiley West Seitz, Falk Hildebrand, Ece Kartal, Marco Raffaele Cosenza, Vladimir Benes, Peer Bork, Christoph Merten. 2021-06-02. Enrichment of gut microbiome strains for cultivation-free genome sequencing using droplet microfluidics. https://doi.org/10.1016/j.crmeth.2021.100137

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