不同DNA聚合酶对运用ARTIC工作流的新型冠状病毒纳米孔测序的影响 |
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引用本文: | 李瑾慧,李沛翰,林彦锋,王凯英,李利忠,邱少富,李 鹏,宋宏彬,贾雷立.不同DNA聚合酶对运用ARTIC工作流的新型冠状病毒纳米孔测序的影响[J].现代生物医学进展,2021(13):2401-2405. |
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作者姓名: | 李瑾慧 李沛翰 林彦锋 王凯英 李利忠 邱少富 李 鹏 宋宏彬 贾雷立 |
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作者单位: | 中国人民解放军疾病预防控制中心 北京 100071;中国人民解放军疾病预防控制中心 北京 100071;军事科学院军事医学研究院 北京 100039 |
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基金项目: | 国家科技重大专项项目(2018ZX10305410-004) |
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摘 要: | 摘要 目的:为了验证不同高保真DNA聚合酶是否会对运用ARTIC工作流进行新型冠状病毒纳米孔测序产生影响。方法:使用英国Nanopore公司MinION测序仪对2份已获得全基因组序列的新冠肺炎确诊病例核酸样本分别采用KAPA HiFi HotStart ReadyMix,PrimeSTAR?誖GXL DNA Polymerase和NEBNext High-Fidelity 2X PCR Master Mix进行ARTIC工作流的多重PCR扩增,对扩增产物进行测序,并对测序质量进行分析。结果:不同高保真DNA聚合酶在相同扩增条件下,扩增产物的质检结果和测序质量均不相同,NEBNext High-Fidelity 2X PCR Master Mix在覆盖度和测序深度上明显好于另外两种酶。结论:NEBNext High-Fidelity 2X PCR Master Mix在纳米孔新型冠状病毒ARTIC快速测序工作流中的应用效果较好。
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关 键 词: | 新型冠状病毒 高保真DNA聚合酶 纳米孔测序 ARTIC工作流 |
收稿时间: | 2020/12/31 0:00:00 |
修稿时间: | 2021/1/27 0:00:00 |
The Influence of Different DNA Polymerases on the Novel Coronavirus Nanopore Sequencing with ARTIC Workflow |
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Abstract: | ABSTRACT Objective: To verify whether the selection of different high-fidelity DNA polymerases has an impact on the results of nanopore sequencing of SARS-CoV-2 with ARTIC-nCoV network workflow. Methods: Multiplex PCR amplification of ARTIC-nCoV network workflow were performed on nucleic acid samples of two confirmed COVID-19 patients with KAPA HiFi HotStart ReadyMix, PrimeSTAR? GXL DNA Polymerase or NEBNext High-Fidelity 2X PCR Master Mix. The amplicons were sequenced on Nanopore MinION platform, then were analyzed. Results: Under same conditions, different high-fidelity DNA polymerases lead to different results of quality inspection and sequencing quality. NEBNext High-Fidelity 2X PCR Master Mix is the best among the three enzymes in coverage and sequencing depth. Conclusion: NEBNext High-Fidelity 2X PCR Master Mix has better effect in nanopore sequencing with ARTIC-nCoV network workflow. |
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Keywords: | SARS-CoV-2 High-fidelity DNA polymerases Nanopore sequencing ARTIC-nCoV network workflow |
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