Hasan Md Shazid, Rahman M Shaminur, Das Prosanto Kumar, Ul Alam A S M Rubayet, Islam Ovinu Kibria, Al-Emran Hassan M, Hossain M Anwar, Jahid Iqbal Kabir
Department of Microbiology, Jashore University of Science and Technology, Jashore 7408, Bangladesh.
Department of Biomedical Engineering, Jashore University of Science and Technology, Jashore 7408, Bangladesh.
MethodsX. 2024 Mar 11;12:102646. doi: 10.1016/j.mex.2024.102646. eCollection 2024 Jun.
A thorough understanding of SARS-CoV-2 genetic features is compulsory to track the ongoing pandemic across multiple geographical locations of the world. Thermo Fisher Scientific USA has developed the Ion AmpliSeq SARS-CoV-2 Research Panel for the targeted sequencing of SARS-CoV-2 complete genome with high coverage and lower error rate. In this study an alternative approach of complete genome sequencing has been validated using different commercial sequencing kits to sequence the SARS-CoV-2. Amplification of cDNA with the SARS-CoV-2 primer pool was performed separately using two different master mixes: 2X environmental master mix (EM) and Platinum™ PCR SuperMix High Fidelity master mix (PM) instead of 5X Ion AmpliSeq™ HiFi Mix whereas NEBNext® Fast DNA Library Prep Set for Ion Torrent™ kit was used as an alternative to Ion AmpliSeq Library Kit Plus for other reagents. This study demonstrated a successful procedure to sequence the SARS-CoV-2 whole genome with average ∼2351 depth and 98.1% of total the reads aligned against the reference sequence (SARS-CoV-2, isolate Wuhan-Hu-1, complete genome). Although genome coverage varied, complete genomes were retrieved for both reagent sets with a reduced cost. This study proposed an alternative approach of high throughput sequencing using Ion torrent technology for the sequencing of SARS-CoV-2 in developing countries where sequencing facilities are low. This blended sequencing technique also offers a low cost protocol in developing countries like Bangladesh.
全面了解严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的基因特征对于追踪全球多个地理位置正在发生的疫情至关重要。美国赛默飞世尔科技公司开发了Ion AmpliSeq SARS-CoV-2研究试剂盒，用于对SARS-CoV-2全基因组进行靶向测序，具有高覆盖率和低错误率。在本研究中，已使用不同的商业测序试剂盒验证了一种全基因组测序的替代方法，用于对SARS-CoV-2进行测序。使用两种不同的预混液分别用SARS-CoV-2引物池扩增cDNA：2X环境预混液（EM）和Platinum™ PCR超保真预混液（PM），而不是5X Ion AmpliSeq™ HiFi Mix，同时使用NEBNext® Fast DNA Library Prep Set for Ion Torrent™试剂盒替代Ion AmpliSeq Library Kit Plus用于其他试剂。本研究展示了一种成功的程序，可对SARS-CoV-2全基因组进行测序，平均深度约为2351，98.1%的总读数与参考序列（SARS-CoV-2，分离株武汉-Hu-1，全基因组）比对。尽管基因组覆盖率有所不同，但两种试剂组均检索到了完整的基因组，且成本降低。本研究提出了一种在测序设施较少的发展中国家使用Ion torrent技术对SARS-CoV-2进行测序的高通量测序替代方法。这种混合测序技术也为孟加拉国等发展中国家提供了一种低成本方案。
