仆从
纳米孔测序
16S核糖体RNA
生物
放大器
计算生物学
DNA测序器
核糖体RNA
基因
细菌基因组大小
聚合酶链反应
DNA测序
细菌细胞结构
遗传学
细菌
基因组
作者
Shinichi Kai,Yoshiyuki Matsuo,So Nakagawa,Kirill Kryukov,Shino Matsukawa,Hiromasa Tanaka,Teppei Iwai,Tadashi Imanishi,Kiichi Hirota
出处
期刊:FEBS Open Bio
[Wiley]
日期:2019-01-29
卷期号:9 (3): 548-557
被引量:97
标识
DOI:10.1002/2211-5463.12590
摘要
Rapid identification of bacterial pathogens is crucial for appropriate and adequate antibiotic treatment, which significantly improves patient outcomes. 16S ribosomal RNA (rRNA) gene amplicon sequencing has proven to be a powerful strategy for diagnosing bacterial infections. We have recently established a sequencing method and bioinformatics pipeline for 16S rRNA gene analysis utilizing the Oxford Nanopore Technologies MinION™ sequencer. In combination with our taxonomy annotation analysis pipeline, the system enabled the molecular detection of bacterial DNA in a reasonable time frame for diagnostic purposes. However, purification of bacterial DNA from specimens remains a rate-limiting step in the workflow. To further accelerate the process of sample preparation, we adopted a direct PCR strategy that amplifies 16S rRNA genes from bacterial cell suspensions without DNA purification. Our results indicate that differences in cell wall morphology significantly affect direct PCR efficiency and sequencing data. Notably, mechanical cell disruption preceding direct PCR was indispensable for obtaining an accurate representation of the specimen bacterial composition. Furthermore, 16S rRNA gene analysis of mock polymicrobial samples indicated that primer sequence optimization is required to avoid preferential detection of particular taxa and to cover a broad range of bacterial species. This study establishes a relatively simple workflow for rapid bacterial identification via MinION™ sequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings.
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