Laboratory of Clinical Virology, Reasearch Laboratory Viruses Vectors and Hosts (LR20-IPT10), Institut Pasteur, University of Tunis-El Manar, Tunis, Tunisia.
Laboratory of Bioinformatics, Biomathematics and Biostatistics (BIMS), Institut Pasteur de Tunisgrid.418517.e (IPT), University of Tunis-El Manar, Tunis, Tunisia.
Microbiol Spectr. 2021 Dec 22;9(3):e0063921. doi: 10.1128/Spectrum.00639-21. Epub 2021 Nov 10.
Recent efforts have reported numerous variants that influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral characteristics, including pathogenicity, transmission rate, and detectability by molecular tests. Whole-genome sequencing based on next-generation sequencing technologies is the method of choice to identify all viral variants; however, the resources needed to use these techniques for a representative number of specimens remain limited in many low- and middle-income countries. To decrease sequencing costs, we developed a primer set allowing partial sequences to be generated in the viral S gene, enabling rapid detection of numerous variants of concern (VOCs) and variants of interest (VOIs); whole-genome sequencing is then performed on a selection of viruses based on partial sequencing results. Two hundred one nasopharyngeal specimens collected during the decreasing phase of a high-transmission COVID-19 wave in Tunisia were analyzed. The results reveal high genetic variability within the sequenced fragment and allow the detection of first introductions in the country of already-known VOCs and VOIs, as well as other variants that have interesting genomic mutations and need to be kept under surveillance. The method of choice for SARS-CoV-2 variant detection is whole-genome sequencing using next-generation sequencing (NGS) technologies. Resources for this technology remain limited in many low- and middle-income countries, where it is not possible to perform whole-genome sequencing for representative numbers of SARS-CoV-2-positive cases. In the present work, we developed a novel strategy based on a first partial Sanger screening in the S gene, which includes key mutations of the already known VOCs and VOIs, for rapid identification of these VOCs and VOIs and to help better select specimens that need to be sequenced by NGS technologies. The second step consists of whole-genome sequencing to allow a holistic view of all variants within the selected viral strains and confirm the initial classification of the strains based on partial S gene sequencing.
最近的研究报告了许多影响严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）病毒特性的变体，包括致病性、传播率和分子检测的可检测性。基于下一代测序技术的全基因组测序是识别所有病毒变体的首选方法；然而，在许多低收入和中等收入国家，使用这些技术对代表性数量的样本进行测序的资源仍然有限。为了降低测序成本，我们开发了一组引物，允许在病毒 S 基因中生成部分序列，从而能够快速检测到许多关注的变体（VOCs）和感兴趣的变体（VOIs）；然后根据部分测序结果对选定的病毒进行全基因组测序。对突尼斯 COVID-19 高传播波下降阶段收集的 201 份鼻咽标本进行了分析。结果显示，在测序片段内存在高度遗传变异性，并且能够检测到已经在该国引入的已知 VOCs 和 VOIs，以及其他具有有趣基因组突变且需要进行监测的变体。SARS-CoV-2 变体检测的首选方法是使用下一代测序（NGS）技术进行全基因组测序。许多低收入和中等收入国家的资源仍然有限，无法对代表性数量的 SARS-CoV-2 阳性病例进行全基因组测序。在本工作中，我们开发了一种新策略，基于 S 基因中的第一个部分 Sanger 筛选，该筛选包括已经已知的 VOCs 和 VOIs 的关键突变，用于快速识别这些 VOCs 和 VOIs，并帮助更好地选择需要通过 NGS 技术进行测序的样本。第二步是进行全基因组测序，以允许对选定病毒株内的所有变体进行全面观察，并根据部分 S 基因测序确认初始分类。
