Dataset Information

A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses.

ABSTRACT: The diseases caused by capripoxviruses (CaPVs) are of major economic concern in sheep, goat and cattle as they are inexorably spreading into non-endemic regions. As CaPV strains are serologically indistinguishable and genetically highly homologous, typing closely related strains can only be achieved by whole genome sequencing. Unfortunately the number of publicly available genomes remains low as most sequencing methods rely on virus isolation. Therefore, we developed a robust, cost-effective and widely applicable method that allows to generate (nearly) complete CaPV genomes directly from clinical samples or commercial vaccine batches. A set of pan-CaPVs long-range PCRs spanning the entire genome was designed to generate PCR amplicons that can be sequenced on commonly used high-throughput sequencing platforms: MiSeq (Illumina), RSII (PacBio) and MinION (Oxford Nanopore Technologies). The robustness of the LR-PCR strategy was evaluated for all 3 members of CaPV directly from a variety of samples, including clinical samples (N = 7), vaccine batches (N = 6), and virus isolates (N = 2). The sequencing method described here allows to reconstruct (nearly) complete CaPV genomes in less than a week and will aid researchers studying closely-related CaPV strains worldwide.

SUBMITTER: Mathijs E

PROVIDER: S-EPMC8872832 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Publications

A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses.

Mathijs Elisabeth E Haegeman Andy A De Clercq Kris K Van Borm Steven S Vandenbussche Frank F

Journal of virological methods 20220113

The diseases caused by capripoxviruses (CaPVs) are of major economic concern in sheep, goat and cattle as they are inexorably spreading into non-endemic regions. As CaPV strains are serologically indistinguishable and genetically highly homologous, typing closely related strains can only be achieved by whole genome sequencing. Unfortunately the number of publicly available genomes remains low as most sequencing methods rely on virus isolation. Therefore, we developed a robust, cost-effective and ...[more]

PMID: 35032481

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Project description:Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections causing significant morbidity and mortality among children and the elderly; two RSV vaccines and a monoclonal antibody have recently been approved. Thus, there is an increasing need for a detailed and continuous genomic surveillance of RSV circulating in resource-rich and resource-limited settings worldwide. However, robust, cost-effective methods for whole genome sequencing of RSV from clinical samples that are amenable to high-throughput are still scarce. We developed Next-RSV-SEQ, an experimental and computational pipeline to generate whole genome sequences of historic and current RSV genotypes by in-solution hybridization capture-based next generation sequencing. We optimized this workflow by automating library preparation and pooling libraries prior to enrichment in order to reduce hands-on time and cost, thereby augmenting scalability. Next-RSV-SEQ yielded near-complete to complete genome sequences for 98% of specimens with Cp values ≤31, at median on-target reads >93%, and mean coverage depths between ~1,000 and >5,000, depending on viral load. Whole genomes were successfully recovered from samples with viral loads as low as 230 copies per microliter RNA. We demonstrate that the method can be expanded to other respiratory viruses like parainfluenza virus and human metapneumovirus. Next-RSV-SEQ produces high-quality RSV genomes directly from culture isolates and, more importantly, clinical specimens of all genotypes in circulation. It is cost-efficient, scalable, and can be extended to other respiratory viruses, thereby opening new perspectives for a future effective and broad genomic surveillance of respiratory viruses.ImportanceRespiratory syncytial virus (RSV) is a leading cause of severe acute respiratory tract infections in children and the elderly, and its prevention has become an increasing priority. Recently, vaccines and a long-acting monoclonal antibody to protect effectively against severe disease have been approved for the first time. Hence, there is an urgent need for genomic surveillance of RSV at the global scale to monitor virus evolution, especially with an eye toward immune evasion. However, robust, cost-effective methods for RSV whole genome sequencing that are suitable for high-throughput of clinical samples are currently scarce. Therefore, we have developed Next-RSV-SEQ, an experimental and computational pipeline that produces reliably high-quality RSV genomes directly from clinical specimens and isolates.

Dataset Information

A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses.

Publications

A robust, cost-effective and widely applicable whole-genome sequencing protocol for capripoxviruses.

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