In the Czech Republic, the current pandemic led to over 1.67 million SARS-CoV-2- positive cases since the recording of the first case on 1 March 2020. SARS-CoV-2 genome analysis is an important tool for effective real-time quantitative PCR (RT-qPCR) diagnostics, epidemiology monitoring, as well as vaccination strategy. To date, there is no comprehensive report on the distribution of SARS-CoV-2 genome variants in either the Czech Republic, including Central and Eastern Europe in general, during the first year of pandemic. In this study, we have analysed a representative cohort of SARS-CoV-2 genomes from 229 nasopharyngeal swabs of COVID-19 positive patients collected between March 2020 and February 2021 using validated reference-based sequencing workflow. We document the changing frequency of dominant variants of SARS-CoV-2 (from B.1 -> B.1.1.266 -> B.1.258 -> B.1.1.7) throughout the first year of the pandemic and list specific variants that could impact the diagnostic efficiency RT-qPCR assays. Moreover, our reference-based workflow provided evidence of superinfection in several samples, which may have contributed to one of the highest per capita numbers of COVID-19 cases and deaths during the first year of the pandemic in the Czech Republic.

Center of Molecular Biology and Genetics Department of Internal Medicine Haematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Černopolní 212 9 625 00 Brno Czech Republic

Department of Biology and Medical Genetics 2nd Faculty of Medicine Charles University and Motol University Hospital 5 Úvalu 84 150 05 Prague Czech Republic

Department of Genetics and Microbiology Faculty of Science Charles University Albertov 6 128 00 Prague Czech Republic

Department of Immunology Faculty of Medicine and Dentistry Palacký University Olomouc and University Hospital Hněvotínská 3 775 15 Olomouc Czech Republic

Department of Medical Microbiology 2nd Faculty of Medicine Charles University and Motol University Hospital 5 Úvalu 84 150 06 Prague Czech Republic

Department of Parasitology BIOCEV Faculty of Science Charles University Průmyslová 595 252 50 Vestec Czech Republic

Institute of Applied Biotechnologies Služeb 3056 4 108 00 Prague Czech Republic

Laboratory of Experimental Medicine Institute of Molecular and Translational Medicine Faculty of Medicine and Dentistry Palacky University Olomouc and University Hospital Olomouc Hněvotínská 5 77 515 Olomouc Czech Republic

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