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      Virome Capture Sequencing Enables Sensitive Viral Diagnosis and Comprehensive Virome Analysis

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          ABSTRACT 

          Insensitivity and technical complexity have impeded the implementation of high-throughput nucleic acid sequencing in differential diagnosis of viral infections in clinical laboratories. Here, we describe the development of a virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) that increases the sensitivity of sequence-based virus detection and characterization. The system uses ~2 million probes that cover the genomes of members of the 207 viral taxa known to infect vertebrates, including humans. A biotinylated oligonucleotide library was synthesized on the NimbleGen cleavable array platform and used for solution-based capture of viral nucleic acids present in complex samples containing variable proportions of viral and host nucleic acids. The use of VirCapSeq-VERT resulted in a 100- to 10,000-fold increase in viral reads from blood and tissue homogenates compared to conventional Illumina sequencing using established virus enrichment procedures, including filtration, nuclease treatments, and RiboZero rRNA subtraction. VirCapSeq-VERT had a limit of detection comparable to that of agent-specific real-time PCR in serum, blood, and tissue extracts. Furthermore, the method identified novel viruses whose genomes were approximately 40% different from the known virus genomes used for designing the probe library. The VirCapSeq-VERT platform is ideally suited for analyses of virome composition and dynamics.

          Importance  VirCapSeq-VERT enables detection of viral sequences in complex sample backgrounds, including those found in clinical specimens, such as serum, blood, and tissue. The highly multiplexed nature of the system allows both the simultaneous identification and the comprehensive genetic characterization of all known vertebrate viruses, their genetic variants, and novel viruses. The operational simplicity and efficiency of the VirCapSeq-VERT platform may facilitate transition of high-throughput sequencing to clinical diagnostic as well as research applications.

          Importance 

          VirCapSeq-VERT enables detection of viral sequences in complex sample backgrounds, including those found in clinical specimens, such as serum, blood, and tissue. The highly multiplexed nature of the system allows both the simultaneous identification and the comprehensive genetic characterization of all known vertebrate viruses, their genetic variants, and novel viruses. The operational simplicity and efficiency of the VirCapSeq-VERT platform may facilitate transition of high-throughput sequencing to clinical diagnostic as well as research applications.

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          Most cited references29

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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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            Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome.

            A random-primed complementary DNA library was constructed from plasma containing the uncharacterized non-A, non-B hepatitis (NANBH) agent and screened with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents.
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              Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction

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                Author and article information

                Journal
                mBio
                MBio
                mbio
                mbio
                mBio
                mBio
                American Society of Microbiology (1752 N St., N.W., Washington, DC )
                2150-7511
                22 September 2015
                Sep-Oct 2015
                : 6
                : 5
                : e01491-15
                Affiliations
                [a ]Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, New York, USA
                [b ]Department of Epidemiology, Mailman School of Public Columbia University, New York, New York, USA
                [c ]Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
                [d ]Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
                Author notes
                Address correspondence to W. Ian Lipkin, wil2001@ 123456columbia.edu .

                T. Briese and A. Kapoor contributed equally to this work.

                Editor Anne Moscona, Weill Medical College-Cornell

                This article is a direct contribution from a Fellow of the American Academy of Microbiology.

                Article
                mBio01491-15
                10.1128/mBio.01491-15
                4611031
                26396248
                030adfca-7794-47db-ba34-70d4d0a765c4
                Copyright © 2015 Briese et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 8 September 2015
                : 10 September 2015
                Page count
                supplementary-material: 10, Figures: 6, Tables: 5, Equations: 0, References: 42, Pages: 11, Words: 8173
                Categories
                Research Article
                Custom metadata
                September/October 2015

                Life sciences
                Life sciences

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