Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with PCR-tiling approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, including human and bacterial contamination, as well as the presence of reads derived from the viral sub-genomic RNAs.
Background The genomes of SARS-CoV-2 are classified into variants, some of which are monitored as variants of concern (e.g. the Delta variant B.1.617.2 or Omicron variant B.1.1.529). Proportions of these variants circulating in a human population are typically estimated by large-scale sequencing of individual patient samples. Sequencing a mixture of SARS-CoV-2 RNA molecules from wastewater provides a cost-effective alternative, but requires methods for estimating variant proportions in a mixed sample. Results We propose a new method based on a probabilistic model of sequencing reads, capturing sequence diversity present within individual variants, as well as sequencing errors. The algorithm is implemented in an open source Python program called VirPool. We evaluate the accuracy of VirPool on several simulated and real sequencing data sets from both Illumina and nanopore sequencing platforms, including wastewater samples from Austria and France monitoring the onset of the Alpha variant. Conclusions VirPool is a versatile tool for wastewater and other mixed-sample analysis that can handle both short- and long-read sequencing data. Our approach does not require pre-selection of characteristic mutations for variant profiles, it is able to use the entire length of reads instead of just the most informative positions, and can also capture haplotype dependencies within a single read.
Surveillance of the SARS-CoV-2 variants including the quickly spreading mutants by rapid and near real-time sequencing of the viral genome provides an important tool for effective health policy decision making in the ongoing COVID-19 pandemic. Here we evaluated PCR-tiling of short (~400-bp) and long (~2 and ~2.5-kb) amplicons combined with nanopore sequencing on a MinION device for analysis of the SARS-CoV-2 genome sequences. Analysis of several sequencing runs demonstrated that using the long amplicon schemes outperforms the original protocol based on the 400-bp amplicons. It also illustrated common artefacts and problems associated with this approach, such as uneven genome coverage, variable fraction of discarded sequencing reads, as well as the reads derived from the viral sub-genomic RNAs and/or human and bacterial contamination.
Background: The genomes of SARS-CoV-2 are classified into variants, some of which are monitored as variants of concern (e.g. the delta variant B.1.617.2 or omicron variant B.1.1.529). Proportions of these variants in a population are typically estimated by large-scale sequencing of individual patient samples. Sequencing a mixture of SARS-CoV-2 RNA molecules from wastewater provides a cost-effective alternative, but requires methods for estimating variant proportions in a mixed sample. Results: We propose a new method based on a probabilistic model of sequencing reads, capturing sequence diversity present within individual variants, as well as sequencing errors. The algorithm is implemented in an open source Python program called VirPool. We evaluated the accuracy of VirPool on several simulated and real sequencing data sets from both Illumina and nanopore sequencing platforms, including wastewater samples from Austria and France monitoring the onset of alpha and delta variants. Conclusions: VirPool is a versatile tool for wastewater and other mixed-sample analysis that can handle both short- and long-read sequencing data. Our approach does not require pre-selection of characteristic mutations for variant profiles, it is able to use the entire length of reads instead of just the most informative positions, and can also capture haplotype dependencies within a single read.
The work examines the formation and development of the institution of constitutional control in the Republic of Uzbekistan. The relevance of the study is due to the fact that it allows to analyze the current state of constitutional control expansively, to comprehend the existing problems and to develop proposals for improving the mechanisms of administration of constitutional justice. There is a need to determine promising directions for the development of constitutional control, in the context of the forthcoming growth of the competence of the Constitutional Court and the introduction of the institution of constitutional complaint. The Constitutional Court was created in the history of humankind with the aim of reliable protection of constitutional human rights and freedoms, mutual limitation and balance of interests between the branches of government, ensuring the supremacy of the Constitution and its special protection. The activities of the Constitutional Court of the Republic of Uzbekistan are aimed at protecting the Constitution, ensuring its norms and principles, as well as the human rights and freedoms enshrined in it. It also serves to democratize society and strengthen constitutional legitimacy through improved constitutional review. Over the past period, a solid legal basis has been created for the implementation of constitutional control, improvement of the implementation of constitutional justice, and an increase in the efficiency of constitutional justice.
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