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  • 1
    Online Resource
    Online Resource
    Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism
    MDPI AG ; 2020
    In:  Viruses Vol. 12, No. 10 ( 2020-10-14), p. 1164-
    Details
    In: Viruses, MDPI AG, Vol. 12, No. 10 ( 2020-10-14), p. 1164-
    Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.
    Type of Medium: Online Resource
    ISSN: 1999-4915
    URL: Article
    DOI: 10.3390/v12101164
    Language: English
    Publisher: MDPI AG
    Publication Date: 2020
    detail.hit.zdb_id: 2516098-9
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  • 2
    Online Resource
    Online Resource
    Detection and Typing of Human Enteroviruses from Clinical Samples by Entire-Capsid Next Generation Sequencing
    MDPI AG ; 2021
    In:  Viruses Vol. 13, No. 4 ( 2021-04-08), p. 641-
    Details
    In: Viruses, MDPI AG, Vol. 13, No. 4 ( 2021-04-08), p. 641-
    Abstract: There are increasing concerns of infections by enteroviruses (EVs) causing severe disease in humans. EV diagnostic laboratory methods show differences in sensitivity and specificity as well as the level of genetic information provided. We examined a detection method for EVs based on next generation sequencing (NGS) analysis of amplicons covering the entire capsid coding region directly synthesized from clinical samples. One hundred and twelve clinical samples from England; previously shown to be positive for EVs, were analyzed. There was high concordance between the results obtained by the new NGS approach and those from the conventional Sanger method used originally with agreement in the serotypes identified in the 83 samples that were typed by both methods. The sensitivity and specificity of the NGS method compared to those of the conventional Sanger sequencing typing assay were 94.74% (95% confidence interval, 73.97% to 99.87%) and 97.85% (92.45% to 99.74%) for Enterovirus A, 93.75% (82.80% to 98.69%) and 89.06% (78.75% to 95.49%) for Enterovirus B, 100% (59.04% to 100%) and 98.10% (93.29% to 99.77%) for Enterovirus C, and 100% (75.29% to 100%) and 100% (96.34% to 100%) for Enterovirus D. The NGS method identified five EVs in previously untyped samples as well as additional viruses in some samples, indicating co-infection. This method can be easily expanded to generate whole-genome EV sequences as we show here for EV-D68. Information from capsid and whole-genome sequences is critical to help identifying the genetic basis for changes in viral properties and establishing accurate spatial-temporal associations between EV strains of public health relevance.
    Type of Medium: Online Resource
    ISSN: 1999-4915
    URL: Article
    DOI: 10.3390/v13040641
    Language: English
    Publisher: MDPI AG
    Publication Date: 2021
    detail.hit.zdb_id: 2516098-9
    Location Call Number Limitation Availability
    BibTip Others were also interested in ...
    Online Resource
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