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

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.

Automated summary

We present a droplet microfluidic method for directly targeting and sorting individual cells from complex microbiome samples, enabling bulk whole-genome sequencing without the need for cultivation. This approach allows for the recovery of bacteria at low ratios and successful enrichment of specific strains for genome assembly. The encapsulation of microbiome samples in microfluidic droplets and the use of custom molecular probes facilitate the selective enrichment of target strains, addressing the lack of cultivated strains and reference genomes.