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

The Influence of Different DNA Polymerases on the Novel Coronavirus Nanopore Sequencing with ARTIC Workflow

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 Hi Fi HotStart ReadyMix,PrimeSTAR?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。

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 Hi Fi HotStart ReadyMix,PrimeSTAR ? GXL DNA Polymerase or NEBNext High-Fidelity 2 X 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 2 X PCR Master Mix is the best among the three enzymes in coverage and sequencing depth. Conclusion: NEBNext High-Fidelity 2 X PCR Master Mix has better effect in nanopore sequencing with ARTIC-nCoV network workflow.