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      Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

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          Abstract

          Novel DNA sequencing techniques, referred to as “next-generation” sequencing (NGS), provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many applications of NGS in diagnostic virology. NGS techniques have been used for high-throughput whole viral genome sequencing, such as sequencing of new influenza viruses, for detection of viral genome variability and evolution within the host, such as investigation of human immunodeficiency virus and human hepatitis C virus quasispecies, and monitoring of low-abundance antiviral drug-resistance mutations. NGS techniques have been applied to metagenomics-based strategies for the detection of unexpected disease-associated viruses and for the discovery of novel human viruses, including cancer-related viruses. Finally, the human virome in healthy and disease conditions has been described by NGS-based metagenomics.

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          Most cited references100

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          Cloning of a human parvovirus by molecular screening of respiratory tract samples.

          The identification of new virus species is a key issue for the study of infectious disease but is technically very difficult. We developed a system for large-scale molecular virus screening of clinical samples based on host DNA depletion, random PCR amplification, large-scale sequencing, and bioinformatics. The technology was applied to pooled human respiratory tract samples. The first experiments detected seven human virus species without the use of any specific reagent. Among the detected viruses were one coronavirus and one parvovirus, both of which were at that time uncharacterized. The parvovirus, provisionally named human bocavirus, was in a retrospective clinical study detected in 17 additional patients and associated with lower respiratory tract infections in children. The molecular virus screening procedure provides a general culture-independent solution to the problem of detecting unknown virus species in single or pooled samples. We suggest that a systematic exploration of the viruses that infect humans, "the human virome," can be initiated.
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            Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.

            Representational difference analysis was used to isolate unique sequences present in more than 90 percent of Kaposi's sarcoma (KS) tissues obtained from patients with acquired immunodeficiency syndrome (AIDS). These sequences were not present in tissue DNA from non-AIDS patients, but were present in 15 percent of non-KS tissue DNA samples from AIDS patients. The sequences are homologous to, but distinct from, capsid and tegument protein genes of the Gammaherpesvirinae, herpesvirus saimiri and Epstein-Barr virus. These KS-associated herpesvirus-like (KSHV) sequences appear to define a new human herpesvirus.
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              Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling circle amplification.

              We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and phi29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications.
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                Author and article information

                Journal
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                Molecular Diversity Preservation International (MDPI)
                1422-0067
                2011
                14 November 2011
                : 12
                : 11
                : 7861-7884
                Affiliations
                [1 ]Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, I-35121 Padova, Italy; E-Mails: enrico.lavezzo@ 123456unipd.it (E.L.); valentina.militello@ 123456unipd.it (V.M.); giorgio.palu@ 123456unipd.it (G.P.)
                [2 ]Department of Biological Chemistry, University of Padova, I-35121 Padova, Italy; E-Mail: stefano.toppo@ 123456unipd.it
                Author notes
                [* ]Author to whom correspondence should be addressed; E-Mail: luisa.barzon@ 123456unipd.it ; Tel.: +39-049-8218946; Fax: +39-049-8272355.
                Article
                ijms-12-07861
                10.3390/ijms12117861
                3233444
                22174638
                3b90de4f-b774-44cd-965d-282df4de246c
                © 2011 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                History
                : 02 September 2011
                : 20 October 2011
                : 07 November 2011
                Categories
                Review

                Molecular biology
                minority variants,next generation sequencing,virome,deep sequencing,drug resistance,virology,molecular diagnosis,metagenomics,human immunodeficiency virus,quasispecies,virus discovery

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