Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2

Changwoo Park; Kwan Woo Kim; Dongju Park; Zohaib Ul Hassan; Edmond Changkyun Park; Chang-Seop Lee; Md Tazikur Rahman; Hana Yi; Seil Kim

doi:10.3389/fmicb.2022.876085

Rapid and sensitive amplicon-based genome sequencing of SARS-CoV-2

Front Microbiol. 2022 Aug 17:13:876085. doi: 10.3389/fmicb.2022.876085. eCollection 2022.

Authors

Changwoo Park^{1

2

3}, Kwan Woo Kim^{4

5}, Dongju Park^{1

2

6}, Zohaib Ul Hassan^{1

2

7}, Edmond Changkyun Park^{2

7

8}, Chang-Seop Lee^{9

10}, Md Tazikur Rahman^{9

11}, Hana Yi^{4

5

12}, Seil Kim^{1

2

7}

Affiliations

¹ Microbiological Analysis Team, Group for Biometrology, Korea Research Institute of Standards and Science (KRISS), Daejeon, South Korea.
² Convergent Research Center for Emerging Virus Infection, Korea Research Institute of Chemical Technology (KRICT), Daejeon, South Korea.
³ Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea.
⁴ Department of Public Health Sciences, Graduate School, Korea University, Seoul, South Korea.
⁵ Interdisciplinary Program in Precision Public Health, Korea University, Seoul, South Korea.
⁶ Department of Biological Science, Chungnam National University College of Bioscience and Biotechnology, Daejeon, South Korea.
⁷ Department of Bio-Analysis Science, University of Science and Technology (UST), Daejeon, South Korea.
⁸ Research Center for Bioconvergence Analysis, Korea Basic Science Institute, Cheongju, South Korea.
⁹ Department of Internal Medicine, Jeonbuk National University Medical School, Jeonju, South Korea.
¹⁰ Research Institute of Clinical Medicine of Jeonbuk National University - Biomedical Research Institute of Jeonbuk National University Hospital, Jeonju, South Korea.
¹¹ Department of Medical Science, Jeonbuk National University Medical School, Jeonju, South Korea.
¹² School of Biosystems and Biomedical Sciences, Korea University, Seoul, South Korea.

Abstract

As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields.

Keywords: MinION; SARS-CoV-2; amplicon-based genome sequencing; long amplicon; reverse transcription digital droplet PCR.