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

TL;DR

This paper introduces a droplet microfluidic technique to isolate and sequence individual microbiome strains directly from complex samples, enabling genome assembly without cultivation and improving access to uncultivated microbial diversity.

Contribution

The study presents a novel droplet microfluidic method for targeted microbiome cell sorting and genome sequencing without cultivation, enhancing strain recovery and genome quality.

Findings

Successfully recovered bacteria at 1:250 ratio from stool samples

Enriched Bacteroides vulgatus for high-quality genome assembly

Protocol effectively removes amplicons and debris for sequencing

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.