Alternative genome sequencing approaches of SARS-CoV-2 using Ion AmpliSeq Technology

Md Shazid Hasan; M Shaminur Rahman; Prosanto Kumar Das; A S M Rubayet Ul Alam; Ovinu Kibria Islam; Hassan M Al-Emran; M Anwar Hossain; Iqbal Kabir Jahid

doi:10.1016/j.mex.2024.102646

Alternative genome sequencing approaches of SARS-CoV-2 using Ion AmpliSeq Technology

MethodsX. 2024 Mar 11:12:102646. doi: 10.1016/j.mex.2024.102646. eCollection 2024 Jun.

Authors

Md Shazid Hasan¹, M Shaminur Rahman¹, Prosanto Kumar Das¹, A S M Rubayet Ul Alam¹, Ovinu Kibria Islam¹, Hassan M Al-Emran², M Anwar Hossain³, Iqbal Kabir Jahid^{1

3}

Affiliations

¹ Department of Microbiology, Jashore University of Science and Technology, Jashore 7408, Bangladesh.
² Department of Biomedical Engineering, Jashore University of Science and Technology, Jashore 7408, Bangladesh.
³ Genome Centre, Jashore University of Science and Technology, Jashore 7408, Bangladesh.

Abstract

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.

Keywords: Alternative approaches; Alternative approaches of SARS-CoV-2 complete genome sequencing; Ion AmpliSeq SARS-CoV-2 Research Panel; Ion torrent technology; NEBNext® Fast DNA Library Prep Set.