Pryszlak, AnnaWenzel, TobiasSeitz, Kiley WestHildebrand, FalkKartal, EceCosenza, Marco RaffaeleBenes, VladimirBork, PeerMerten, Christoph A.2022-01-032022-01-032022-01-032022-01-2410.1016/j.crmeth.2021.100137https://infoscience.epfl.ch/handle/20.500.14299/184281We 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.droplet microfluidicssingle-cell analysisgenomicsgenomegut microbiomefunctional analysismetagenomicsbacteriatext::journal::journal article::research article