使用液滴微流体富集用于无培养基因组测序的肠道微生物组菌株
Enrichment of gut microbiome strains for cultivation-free genome sequencing using droplet microfluidics
摘要：
我们报告了一种液滴微流控方法，用于直接从复杂的微生物组样品中靶向和分选单个细胞，并制备这些细胞用于批量全基因组测序而无需培养。我们通过回收以低至1：250的比例掺入人粪便样品中的细菌，并通过成功地将内源性普通拟杆菌富集到高质量基因组从头组装所需的水平，来表征这种方法。虽然微生物组菌株越来越多地用于生物医学应用，但绝大多数物种和菌株都是未培养的，没有参考基因组。我们通过将复杂的微生物组样品直接封装到微流体液滴中并使用定制的分子TaqMan探针扩增靶特异性基因组片段来解决这一缺点。我们通过液滴分选分离那些阳性液滴，选择性地富集单个靶菌株细胞。最后，我们提出了一个协议，以纯化基因组DNA，同时专门去除扩增子和细胞碎片的高质量的基因组测序。
英文摘要：
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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