Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding
Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding
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DOI：
10.1093/biomethods/bpaa014
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发表时间：
2020-01-01
影响因子：
3.6
通讯作者：
Silander, Olin K.
中科院分区：
文献类型：
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作者：
Freed, Nikki E.;Vlkova, Marketa;Silander, Olin K.
对导致2019年冠状病毒病的严重急性呼吸综合征冠状病毒2（SARS-CoV-2）进行快速且具有成本效益的全基因组测序对于了解病毒传播动力学至关重要。在这里，我们表明，使用一组新的多重引物与牛津纳米孔快速条形码库试剂盒结合，可以更快，更简单，更便宜的SARS-CoV-2基因组测序。与其他常用引物组相比，该引物组产生的扩增子在覆盖度方面表现出较低水平的变化。使用5个C-q值在20和31之间的SARS-CoV-2患者样本，我们证明了高质量的基因组可以用10000个读数（类似于5 Mbp的序列数据）生成。我们还表明，条形码的错误分类，这可能是更有可能使用牛津纳米孔快速条形码库准备时，是不太可能导致变异调用的问题。与更标准的基于连接的Oxford Nanopore文库制备方法相比，这种方法将从RNA到基因组序列的时间减少了一半以上，成本也大大降低。
Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with C-q values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (similar to 5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.