Biomedical Informatics Laboratory, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193, Japan.
Division of Pulmonary Medicine, Department of Medicine, Tokai University School of Medicine, Isehara, Kanagawa, 259-1193, Japan.
Sci Rep. 2017 Jul 18;7(1):5657. doi: 10.1038/s41598-017-05772-5.
We developed a portable system for 16S rDNA analyses consisting of a nanopore technology-based sequencer, the MinION, and laptop computers, and assessed its potential ability to determine bacterial compositions rapidly. We tested our protocols using a mock bacterial community that contained equimolar 16S rDNA and a pleural effusion from a patient with empyema, for time effectiveness and accuracy. MinION sequencing targeting 16S rDNA detected all 20 of the bacterial species present in the mock bacterial community. Time course analysis indicated that the sequence data obtained during the first 5 minutes of sequencing (1,379 bacterial reads) were enough to detect all 20 bacteria in the mock sample and to determine species composition, consistent with results of those obtained from 4 hours of sequencing (24,202 reads). Additionally, using a clinical sample extracted from the empyema patient's pleural effusion, we could identify major bacterial pathogens in that effusion using our rapid sequencing and analysis protocol. All results are comparable to conventional 16S rDNA sequencing results using an IonPGM sequencer. Our results suggest that rapid sequencing and bacterial composition determination are possible within 2 hours after obtaining a DNA sample.
我们开发了一种由基于纳米孔技术的测序仪 MinION 和笔记本电脑组成的便携式 16S rDNA 分析系统，并评估了其快速确定细菌组成的潜在能力。我们使用含有等量 16S rDNA 的模拟细菌群落和脓胸患者的胸腔积液来测试我们的方案，以评估其时间有效性和准确性。针对 16S rDNA 的 MinION 测序检测到模拟细菌群落中存在的所有 20 种细菌。时间过程分析表明，在测序的前 5 分钟（1379 个细菌读段）获得的序列数据足以检测到模拟样本中的所有 20 种细菌，并确定物种组成，这与从 4 小时测序（24202 个读段）中获得的结果一致。此外，使用从脓胸患者胸腔积液中提取的临床样本，我们可以使用我们的快速测序和分析方案来鉴定该积液中的主要细菌病原体。所有结果均与使用 IonPGM 测序仪进行的常规 16S rDNA 测序结果相当。我们的结果表明，在获得 DNA 样本后 2 小时内即可进行快速测序和细菌组成确定。
