Exercising the Sanger Sequencing Strategy for Variants Screening and Full-Length Genome of SARS-CoV-2 Virus during Alpha, Delta, and Omicron Outbreaks in Hiroshima
Ko Ko,
Kazuaki Takahashi,
Shintaro Nagashima,
Bunthen E,
Serge Ouoba,
Toshiro Takafuta,
Yoshiki Fujii,
Michi Mimori,
Fumie Okada,
Eisaku Kishita,
Kunie Ariyoshi,
Md Razeen Ashraf Hussain,
Aya Sugiyama,
Tomoyuki Akita,
Masao Kuwabara,
Junko Tanaka
Affiliations
Ko Ko
Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Kazuaki Takahashi
Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Shintaro Nagashima
Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Bunthen E
Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Serge Ouoba
Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Toshiro Takafuta
Hiroshima City Funairi Citizens Hospital, 14-11, Funairisaiwaicho, Naka-ku, Hiroshima 730-0844, Japan
Yoshiki Fujii
Hiroshima City Institute of Public Health, 4 Chome-1-2, Shoko Center, Nishi-ku, Hiroshima 733-0833, Japan
Michi Mimori
Hiroshima City Health and Welfare Bureau, 1 Chome-6-34, Kokutaijimachi, Naka-ku, Hiroshima 734-0042, Japan
Fumie Okada
Hiroshima Prefectural Health and Welfare Bureau, 10-52, Motomachi, Naka-ku, Hiroshima 730-8511, Japan
Eisaku Kishita
Hiroshima Prefectural Health and Welfare Bureau, 10-52, Motomachi, Naka-ku, Hiroshima 730-8511, Japan
Kunie Ariyoshi
Hiroshima Prefectural Technology Research Institute, Public Health and Environment Center, 1 Chome-6-29, Minamimachi, Minami-ku, Hiroshima 734-0007, Japan
Md Razeen Ashraf Hussain
Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Aya Sugiyama
Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Tomoyuki Akita
Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan
Masao Kuwabara
Hiroshima Prefectural Center for Disease Control and Prevention, 10-52, Motomachi, Naka-ku, Hiroshima 730-8511, Japan
Junko Tanaka
Department of Epidemiology, Infectious Disease Control and Prevention, Graduate School of Biomedical and Health Science, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan
This study aimed to exercise the Sanger sequencing strategy for screening of variants among confirmed COVID-19 cases and validate our strategy against NGS strains in Hiroshima retrieved from GISAID. A total of 660 samples from confirmed COVID-19 cases underwent screening for variants by Sanger-based partial sequencing to the targeted spike gene (nt22,735~nt23,532) using an in-house-developed primer set. The identification of variants was done by unique checkpoints of base nucleotide changes in the targeted spike gene. Moreover, we amplified one full-length genome using Sanger method and an in-house-developed primer library. Using NGS strains of the same sampling period from GISAID, a phylogenetic tree was constructed to examine the distribution pattern of variants in Hiroshima and to validate our Sanger method. The modified primer set provided 100% validation and 99.2% amplification. PANGO Lineage R.1 was detected in late in the third wave, followed by Alpha (B.1.1.7) domination in the fourth wave, Delta (B.1.617.2) domination in the fifth wave, and Omicron (B.1.1.529) domination in the sixth wave, and there was no significant difference in viral copies between variants (p = 0.09). The variants showed different transmission patterns, but the distribution of variants is consistent to that shown by the phylogenetic tree. The Sanger method also provided successful amplification of the full-length genome of the SARS-CoV-2 virus. Our Sanger sequencing strategy was useful for the screening of SASR-CoV-2 variants without the need for full-genome amplification. The modified primer set was validated to use universally, which allows an understanding of the variants’ distribution in real time and provides the evidence for policy-making and the formulation or modification of preventive strategies.
