Whole genome sequencing is emerging as a promising tool for the untargeted detection of a broad range of microbial species for diagnosis and analysis. However, it is logistically challenging to perform the multistep process from sample preparation to DNA amplification to sequencing and analysis within a short turnaround time. To address this challenge, we developed a digital microfluidic device for rapid whole genome amplification of low-abundance bacterial DNA and compared results with conventional in-tube DNA amplification. In this work, we chose Corynebacterium glutamicum DNA as a bacterial target for method development and optimization, as it is not a common contaminant. Sequencing was performed in a hand-held Oxford Nanopore Technologies MinION sequencer. Our results show that using an in-tube amplification approach, at least 1 pg starting DNA is needed to reach the amount required for successful sequencing within 2 h. While using a digital microfluidic device, it is possible to amplify as low as 10 fg of C. glutamicum DNA (equivalent to the amount of DNA within a single bacterial cell) within 2 h and to identify the target bacterium within 30 min of MinION sequencing—100× lower than the detection limit of an in-tube amplification approach. We demonstrate the detection of C. glutamicum DNA in a mock community DNA sample and characterize the limit of bacterial detection in the presence of human cells. This approach can be used to identify microbes with minute amounts of genetic material in samples depleted of human cells within 3 h.

中文翻译：

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使用用于 MinION 测序的数字微流体平台在 3 小时内扩增细菌 DNA 的飞克数

全基因组测序正在成为一种有前途的工具，可用于非靶向检测范围广泛的微生物物种以进行诊断和分析。然而，在短时间内执行从样品制备到 DNA 扩增再到测序和分析的多步骤过程在逻辑上具有挑战性。为了应对这一挑战，我们开发了一种数字微流体装置，用于低丰度细菌 DNA 的快速全基因组扩增，并将结果与​​传统的管内 DNA 扩增进行比较。在这项工作中，我们选择了谷氨酸棒杆菌DNA 作为方法开发和优化的细菌目标，因为它不是常见的污染物。测序在手持式 Oxford Nanopore Technologies MinION 测序仪中进行。我们的结果表明，使用管内扩增方法，至少需要 1 pg 起始 DNA 才能在 2 小时内达到成功测序所需的量。使用数字微流体装置时，可以在 2 小时内扩增低至 10 fg 的谷氨酸棒杆菌DNA（相当于单个细菌细胞内的 DNA 量），并在 MinION 测序的 30 分钟内识别目标细菌— 比管内扩增方法的检测限低 100 倍。我们展示了谷氨酸棒杆菌的检测模拟社区 DNA 样本中的 DNA，并表征人类细胞存在下细菌检测的极限。这种方法可用于在 3 小时内耗尽人类细胞的样本中识别具有微量遗传物质的微生物。