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1.
Viruses ; 14(6)2022 06 06.
Article in English | MEDLINE | ID: covidwho-1884383

ABSTRACT

The COVID-19 pandemic demonstrated how rapidly various molecular methods can be adapted for a Public Health Emergency. Whether a need arises for whole-genome studies (next-generation sequencing), fast and high-throughput diagnostics (reverse-transcription real-time PCR) or global immunization (construction of mRNA or viral vector vaccines), the scientific community has been able to answer all these calls. In this study, we aimed at the assessment of effectiveness of the commercially available solution for full-genome SARS-CoV-2 sequencing (AmpliSeq™ SARS-CoV-2 Research Panel and Ion AmpliSeq™ Library Kit Plus, Thermo Fisher Scientific). The study is based on 634 samples obtained from patients from Poland, with varying viral load, assigned to a number of lineages. Here, we also present the results of protocol modifications implemented to obtain high-quality genomic data. We found that a modified library preparation protocol required less viral RNA input in order to obtain the optimal library quantity. Concurrently, neither concentration of cDNA nor reamplification of libraries from low-template samples improved the results of sequencing. On the basis of the amplicon success rates, we propose one amplicon to be redesigned, namely, the r1_1.15.1421280, for which less than 50 reads were produced by 44% of samples. Additionally, we found several mutations within different SARS-CoV-2 lineages that cause the neighboring amplicons to underperform. Therefore, due to constant SARS-CoV-2 evolution, we support the idea of conducting ongoing sequence-based surveillance studies to continuously validate commercially available RT-PCR and whole-genome sequencing solutions.


Subject(s)
COVID-19 , SARS-CoV-2 , Genome, Viral , High-Throughput Nucleotide Sequencing/methods , Humans , Pandemics , SARS-CoV-2/genetics , Technology
2.
Viruses ; 13(7)2021 07 02.
Article in English | MEDLINE | ID: covidwho-1295939

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) evolved into a worldwide outbreak, with the first Polish cases in February/March 2020. This study aimed to investigate the molecular epidemiology of the circulating virus lineages between March 2020 and February 2021. We performed variant identification, spike mutation pattern analysis, and phylogenetic and evolutionary analyses for 1106 high-coverage whole-genome sequences, implementing maximum likelihood, multiple continuous-time Markov chain, and Bayesian birth-death skyline models. For time trends, logistic regression was used. In the dataset, virus B.1.221 lineage was predominant (15.37%), followed by B.1.258 (15.01%) and B.1.1.29 (11.48%) strains. Three clades were identified, being responsible for 74.41% of infections over the analyzed period. Expansion in variant diversity was observed since September 2020 with increasing frequency of the number in spike substitutions, mainly H69V70 deletion, P681H, N439K, and S98F. In population dynamics inferences, three periods with exponential increase in infection were observed, beginning in March, July, and September 2020, respectively, and were driven by different virus clades. Additionally, a notable increase in infections caused by the B.1.1.7 lineage since February 2021 was noted. Over time, the virus accumulated mutations related to optimized transmissibility; therefore, faster dissemination is reflected by the second wave of epidemics in Poland.


Subject(s)
COVID-19/epidemiology , COVID-19/virology , SARS-CoV-2/classification , SARS-CoV-2/genetics , Bayes Theorem , Evolution, Molecular , Genetic Variation , Genome, Viral , Humans , Molecular Epidemiology , Mutation , Phylogeny , Poland/epidemiology , Prevalence , Whole Genome Sequencing
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