Rapid Acquisition of High-Quality SARS-CoV-2 Genome via Amplicon-Oxford Nanopore Sequencing.
Rapid Acquisition of High-Quality SARS-CoV-2 Genome via Amplicon-Oxford Nanopore Sequencing.
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DOI：
10.1007/s12250-021-00378-8
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发表时间：
2021-10
影响因子：
5.5
通讯作者：
Chen J
中科院分区：
文献类型：
--
作者：
Yan Y;Wu K;Chen J;Liu H;Huang Y;Zhang Y;Xiong J;Quan W;Wu X;Liang Y;He K;Jia Z;Wang D;Liu D;Wei H;Chen J
基因组测序在COVID-19大流行的初始阶段显示出强大的能力，如病原体识别和病毒初步追踪。然而，SARS冠状病毒2型基因组的快速获取由于其核酸载量低和核酸背景复杂而受到限制。为了解决这个问题，我们通过利用SARS-CoV-2特异性扩增子扩增和Oxford Nanopore PromethION平台修改和评估了一种方法。该工作流程从COVID-19患者的咽拭子开始，结合反转录PCR，一步多重扩增，缩短实验时间，即可在24 h内快速稳定获得高质量的SARS-CoV-2基因组。对42份样本进行了综合评价：测序质量与样本的病毒载量相关性良好，Ct值达到39.14的样本可稳定获得高质量的SARS-CoV-2基因组，对不同Ct值的数据产量进行了评估，Ct值小于20的样本推荐测序时间为8 h;变异分析表明，该方法也可以检测现有和新出现的基因组突变; Illumina测序验证了超深度测序可以大大改善Nanopore测序的单读错误率，使其低至0.4/10，000 bp。综上所述，利用扩增子扩增技术可以获得高质量的SARS-CoV-2基因组，是加速SARS-CoV-2遗传资源获取和基因组多样性追踪的有效方法。在线版本包含补充材料，可通过10.1007/s12250-021-00378-8获取。
Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing. While the rapid acquisition of SARS-CoV-2 genome from clinical specimens is limited by their low nucleic acid load and the complexity of the nucleic acid background. To address this issue, we modified and evaluated an approach by utilizing SARS-CoV-2-specific amplicon amplification and Oxford Nanopore PromethION platform. This workflow started with the throat swab of the COVID-19 patient, combined reverse transcript PCR, and multi-amplification in one-step to shorten the experiment time, then can quickly and steadily obtain high-quality SARS-CoV-2 genome within 24 h. A comprehensive evaluation of the method was conducted in 42 samples: the sequencing quality of the method was correlated well with the viral load of the samples; high-quality SARS-CoV-2 genome could be obtained stably in the samples with Ct value up to 39.14; data yielding for different Ct values were assessed and the recommended sequencing time was 8 h for samples with Ct value of less than 20; variation analysis indicated that the method can detect the existing and emerging genomic mutations as well; Illumina sequencing verified that ultra-deep sequencing can greatly improve the single read error rate of Nanopore sequencing, making it as low as 0.4/10,000 bp. In summary, high-quality SARS-CoV-2 genome can be acquired by utilizing the amplicon amplification and it is an effective method in accelerating the acquisition of genetic resources and tracking the genome diversity of SARS-CoV-2. The online version contains supplementary material available at 10.1007/s12250-021-00378-8.
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影响因子：
9.5
作者：
Katoh K;Rozewicki J;Yamada KD
通讯作者：
Yamada KD
DOI：
10.1038/nrmicro.2016.81
发表时间：
2016-08
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
de Wit E;van Doremalen N;Falzarano D;Munster VJ
通讯作者：
Munster VJ
影响因子：
168.9
作者：
Huang, Chaolin;Wang, Yeming;Cao, Bin
通讯作者：
Cao, Bin
影响因子：
12.3
作者：
Jain M;Olsen HE;Paten B;Akeson M
通讯作者：
Akeson M
影响因子：
5.8
作者：
Li, Heng
通讯作者：
Li, Heng