Analysis of the ARTIC Version 3 and Version 4 SARS-CoV-2 Primers and Their Impact on the Detection of the G142D Amino Acid Substitution in the Spike Protein.
Analysis of the ARTIC Version 3 and Version 4 SARS-CoV-2 Primers and Their Impact on the Detection of the G142D Amino Acid Substitution in the Spike Protein.
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DOI：
10.1128/spectrum.01803-21
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发表时间：
2021-12-22
影响因子：
3.7
通讯作者：
Musser JM
中科院分区：
文献类型：
--
作者：
Davis JJ;Long SW;Christensen PA;Olsen RJ;Olson R;Shukla M;Subedi S;Stevens R;Musser JM
ARTIC网络提供了PCR引物序列的公共资源和扩增SARS-CoV-2基因组的建议。最初的平铺策略是用参考基因组Wuhan-01开发的，随后的迭代解决了低扩增和序列丢失的区域。最近，基于新的变异基因组序列发布了一个新版本（V4），以响应一些V3引物位于关键突变区域的认识。在此，我们将ARTIC V3和V4引物组的性能与用Illumina NovaSeq 6000仪器测序的663个SARS-CoV-2临床样本的匹配组进行比较。我们观察到测序深度和质量的总体改善，以及导致刺突蛋白D950 N变异的SNP分辨率的提高。重要的是，我们还发现在Delta谱系样品中几乎普遍存在刺突蛋白取代G142 D。由于ARTIC V3引物在Delta变体最初激增期间的先前发布和广泛使用，在公共储存库中保存的早期Delta变体基因组中，G142 D氨基酸取代可能基本上代表性不足。除了ARTIC V4引物组的改进性能外，本研究还说明了引物方案在下游分析中的重要性。重要性ARTIC网络引物通常被世界各地的实验室用于扩增和测序临床样本中存在的SARS-CoV-2。随着新变体的进化和传播，发现V3引物组对几个关键突变的扩增较差。在本报告中，我们比较了V3和V4引物组对一组匹配样本的测序结果。我们发现采用ARTIC V4引物组对于SARS-CoV-2刺突区的准确测序至关重要。缺乏描述所用引物方案的元数据将对下游使用公开的SARS-Cov-2测序读数和组装的基因组产生负面影响。
The ARTIC Network provides a common resource of PCR primer sequences and recommendations for amplifying SARS-CoV-2 genomes. The initial tiling strategy was developed with the reference genome Wuhan-01, and subsequent iterations have addressed areas of low amplification and sequence drop out. Recently, a new version (V4) was released, based on new variant genome sequences, in response to the realization that some V3 primers were located in regions with key mutations. Herein, we compare the performance of the ARTIC V3 and V4 primer sets with a matched set of 663 SARS-CoV-2 clinical samples sequenced with an Illumina NovaSeq 6000 instrument. We observe general improvements in sequencing depth and quality, and improved resolution of the SNP causing the D950N variation in the spike protein. Importantly, we also find nearly universal presence of spike protein substitution G142D in Delta-lineage samples. Due to the prior release and widespread use of the ARTIC V3 primers during the initial surge of the Delta variant, it is likely that the G142D amino acid substitution is substantially underrepresented among early Delta variant genomes deposited in public repositories. In addition to the improved performance of the ARTIC V4 primer set, this study also illustrates the importance of the primer scheme in downstream analyses. IMPORTANCE ARTIC Network primers are commonly used by laboratories worldwide to amplify and sequence SARS-CoV-2 present in clinical samples. As new variants have evolved and spread, it was found that the V3 primer set poorly amplified several key mutations. In this report, we compare the results of sequencing a matched set of samples with the V3 and V4 primer sets. We find that adoption of the ARTIC V4 primer set is critical for accurate sequencing of the SARS-CoV-2 spike region. The absence of metadata describing the primer scheme used will negatively impact the downstream use of publicly available SARS-Cov-2 sequencing reads and assembled genomes.