Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima, 734-8551, Japan.
Hiroshima City Funairi Citizens Hospital, Hiroshima, Japan.
BMC Med Genomics. 2023 Aug 24;16(1):199. doi: 10.1186/s12920-023-01633-5.
This study aimed to compare the performance of Sanger-based SARS-CoV-2 spike gene sequencing and Next Generation Sequencing (NGS)-based full-genome sequencing for variant identification in saliva samples with low viral titer.
Using 241 stocked saliva samples collected from confirmed COVID-19 patients between November 2020 and March 2022 in Hiroshima, SARS-CoV-2 spike gene sequencing (nt22735-nt23532) was performed by nested RT-PCR and Sanger platform using in-house primers. The same samples underwent full-genome sequencing by NGS using Illumina NextSeq2000.
Among 241 samples, 147 were amplified by both the Sanger and the Illumina NextSeq2000 NGS, 86 by Sanger only, and 8 were not amplified at all. The overall amplification rates of Illumina NextSeq2000 NGS and Sanger were 61% and 96.7%, respectively. At low viral titer (< 10 copies/mL), Illumina NextSeq2000 NGS provided 19.2% amplification, while Sanger was 89.7% (p < 0.0001). Both platforms identified 38 wild type, 54 Alpha variants, 84 Delta variants, and 57 Omicron variants.
Our study provided evidence to expand the capacity of Sanger-based SARS-CoV-2 spike gene sequencing for variants identification over full-genome by Illumina NextSeq2000 NGS for mass screening. Therefore, the feasible and simple Sanger-based SARS-CoV-2 spike gene sequencing is practical for the initial variants screening, which might reduce the gap between the rapid evolution of SARS-CoV-2 and its molecular surveillance.
本研究旨在比较基于 Sanger 的 SARS-CoV-2 刺突基因测序和基于下一代测序（NGS）的全基因组测序在低病毒载量唾液样本中用于变异识别的性能。
使用 2020 年 11 月至 2022 年 3 月在广岛收集的 241 份确诊 COVID-19 患者的唾液样本，通过嵌套 RT-PCR 和 Sanger 平台使用内部引物进行 SARS-CoV-2 刺突基因测序（nt22735-nt23532）。相同的样本通过 Illumina NextSeq2000 进行全基因组测序。
在 241 个样本中，147 个样本通过 Sanger 和 Illumina NextSeq2000 NGS 均扩增，86 个样本仅通过 Sanger 扩增，8 个样本根本没有扩增。Illumina NextSeq2000 NGS 和 Sanger 的总体扩增率分别为 61%和 96.7%。在低病毒载量（<10 拷贝/mL）下，Illumina NextSeq2000 NGS 的扩增率为 19.2%，而 Sanger 的扩增率为 89.7%（p<0.0001）。两种平台均鉴定出 38 个野生型、54 个 Alpha 变体、84 个 Delta 变体和 57 个奥密克戎变体。
本研究为在大规模筛查中通过 Illumina NextSeq2000 NGS 扩展基于 Sanger 的 SARS-CoV-2 刺突基因测序对变体识别的能力提供了证据，而不是全基因组测序。因此，可行且简单的基于 Sanger 的 SARS-CoV-2 刺突基因测序对于初始变体筛查是实用的，这可能会缩小 SARS-CoV-2 的快速进化与其分子监测之间的差距。
