Department of Forensic Medicine, Pomeranian Medical University in Szczecin, 70-111 Szczecin, Poland.
Department of Infectious, Tropical Diseases and Immune Deficiency, Pomeranian Medical University in Szczecin, 71-455 Szczecin, Poland.
Viruses. 2022 Jun 6;14(6):1230. doi: 10.3390/v14061230.
The COVID-19 pandemic demonstrated how rapidly various molecular methods can be adapted for a Public Health Emergency. Whether a need arises for whole-genome studies (next-generation sequencing), fast and high-throughput diagnostics (reverse-transcription real-time PCR) or global immunization (construction of mRNA or viral vector vaccines), the scientific community has been able to answer all these calls. In this study, we aimed at the assessment of effectiveness of the commercially available solution for full-genome SARS-CoV-2 sequencing (AmpliSeq™ SARS-CoV-2 Research Panel and Ion AmpliSeq™ Library Kit Plus, Thermo Fisher Scientific). The study is based on 634 samples obtained from patients from Poland, with varying viral load, assigned to a number of lineages. Here, we also present the results of protocol modifications implemented to obtain high-quality genomic data. We found that a modified library preparation protocol required less viral RNA input in order to obtain the optimal library quantity. Concurrently, neither concentration of cDNA nor reamplification of libraries from low-template samples improved the results of sequencing. On the basis of the amplicon success rates, we propose one amplicon to be redesigned, namely, the r1_1.15.1421280, for which less than 50 reads were produced by 44% of samples. Additionally, we found several mutations within different SARS-CoV-2 lineages that cause the neighboring amplicons to underperform. Therefore, due to constant SARS-CoV-2 evolution, we support the idea of conducting ongoing sequence-based surveillance studies to continuously validate commercially available RT-PCR and whole-genome sequencing solutions.
COVID-19 大流行表明，各种分子方法可以多快适应公共卫生紧急情况。无论是需要进行全基因组研究（下一代测序）、快速高通量诊断（逆转录实时 PCR）还是全球免疫接种（mRNA 或病毒载体疫苗的构建），科学界都能够应对所有这些需求。在这项研究中，我们旨在评估市售的用于全基因组 SARS-CoV-2 测序的解决方案（AmpliSeq™ SARS-CoV-2 Research Panel 和 Ion AmpliSeq™ Library Kit Plus，Thermo Fisher Scientific）的有效性。该研究基于从波兰患者中获得的 634 个具有不同病毒载量的样本，这些样本被分配到多个谱系中。在这里，我们还介绍了为获得高质量基因组数据而实施的协议修改的结果。我们发现，修改后的文库制备协议需要较少的病毒 RNA 输入，以获得最佳文库数量。同时，cDNA 的浓度或从低模板样本中再扩增文库都不能改善测序结果。根据扩增子的成功率，我们建议重新设计一个扩增子，即 r1_1.15.1421280，因为 44%的样本中产生的该扩增子的读取数少于 50。此外，我们在不同的 SARS-CoV-2 谱系中发现了几个导致相邻扩增子表现不佳的突变。因此，由于 SARS-CoV-2 的不断进化，我们支持开展基于序列的持续监测研究的想法，以不断验证市售的 RT-PCR 和全基因组测序解决方案。
