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

Abstract

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.

The predictive markers of severity and mortality in hospitalized hemodialysis patients with COVID‐19 during Omicron epidemic

“…We excluded HD patients with COVID‐19 who were treated on an outpatient basis. In December 2021, the Omicron variant (B1.1.529) was detected, and in January 2022, most were infected with the Omicron variant (B1.1.529) (89%), followed by the Delta variant (B1.1.617) (11%) in Hiroshima in Japan [16]. Therefore, the study period coincided with the Omicron epidemic.…”

Variant-specific Symptoms After COVID-19: A Hospital-based Study in Hiroshima

“…Third, we could not test specific SARS-CoV-2 strains in each subject, instead, patients were categorized into groups based on the epidemic periods of each strain as reported in Hiroshima. 19 Fourth, we conducted this study using self-administered questionnaires, which introduces the potential for both underestimation and overestimation of the impact of post-COVID-19 symptoms. Fifth, this study questionnaire did not include an assessment of symptom severity or the impact of post-COVID-19 symptoms on the daily lives of patients.…”

“…To investigate variant-specific symptoms following COVID-19, participants were grouped into four categories based on their symptom onset date and the monthly distribution of the dominant variant in Hiroshima Prefecture. 19 These groups were designated as follows:…”

Targeted Sanger Sequencing of a Cluster of COVID-19 Cases in the Surgical ICU of a Non-COVID Hospital: Lessons Learned

“…Their later study also pointed out that the genomic information coverage among confirmed COVID-19 cases is too low when using NGS. It reiterated the usefulness of Sanger sequencing not just in understanding variant distribution over time but also in providing evidence for policy making and the formulation or modification of preventive strategies [ 14 , 15 ]. In this study, a similar Sanger-based approach was adapted to a much smaller setting, like our non-COVID hospital.…”