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      Use of Unamplified RNA/cDNA–Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses

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          Abstract

          Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization.

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          Most cited references4

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          MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island.

          Short-read, high-throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes. The MinION nanopore sequencer can produce long sequencing reads on a device similar in size to a USB memory stick. Here we apply a MinION sequencer to resolve the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58. Nanopore sequencing data from a single 18-h run was used to create a scaffold for an assembly generated from short-read Illumina data. Our results demonstrate the potential of the MinION device in clinical laboratories to fully characterize the epidemic spread of bacterial pathogens.
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            Is Open Access

            Temporal and spatial analysis of the 2014–2015 Ebola virus outbreak in West Africa

            Analysis of 179 new Ebola virus sequences from patient samples collected in Guinea between March 2014 and January 2015 shows how different lineages evolved and spread in West Africa. Supplementary information The online version of this article (doi:10.1038/nature14594) contains supplementary material, which is available to authorized users. Miles Carroll and colleagues report describe the genetic evolution of Ebola virus circulating in West Africa, based on 179 new virus sequences from patient samples collected in Guinea between March 2014 and January 2015. Their analysis shows how different lineages evolved and spread in West Africa between Sierra Leone, Guinea and Liberia. Supplementary information The online version of this article (doi:10.1038/nature14594) contains supplementary material, which is available to authorized users. West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded 1 , 2 , 3 . Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2 ). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak. Supplementary information The online version of this article (doi:10.1038/nature14594) contains supplementary material, which is available to authorized users.
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              Nanopore Sequencing as a Rapidly Deployable Ebola Outbreak Tool

              Rapid sequencing of RNA/DNA from pathogen samples obtained during disease outbreaks provides critical scientific and public health information. However, challenges exist for exporting samples to laboratories or establishing conventional sequencers in remote outbreak regions. We successfully used a novel, pocket-sized nanopore sequencer at a field diagnostic laboratory in Liberia during the current Ebola virus outbreak.
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                Author and article information

                Journal
                Emerg Infect Dis
                Emerging Infect. Dis
                EID
                Emerging Infectious Diseases
                Centers for Disease Control and Prevention
                1080-6040
                1080-6059
                August 2016
                : 22
                : 8
                : 1448-1451
                Affiliations
                [1]US Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland, USA (A. Kilianski, P.A. Roth, A.T. Liem, J.M. Hill, K.L. Willis, R.D. Rossmaier, A.V. Marinich, M.N. Maughan, M.A. Karavis, C.N. Rosenzweig);
                [2]Defense Threat Reduction Agency, Fort Belvoir, Virginia, USA (K.L. Willis);
                [3]National Institutes of Health, Fort Detrick, Frederick, Maryland, USA (J.H. Kuhn, A.N. Honko)
                Author notes
                Address for correspondence: C. Nicole Rosenzweig BioDefense Branch, BioSciences Division, Edgewood Chemical Biological Center, Aberdeen Proving Ground, MD 21010, USA; email: carolyn.n.rosenzweig.civ@ 123456mail.mil
                Article
                16-0270
                10.3201/eid2208.160270
                4982148
                27191483
                308c49d2-e0e2-4791-b075-0580f561252f
                History
                Categories
                Dispatch
                Dispatch
                Use of Unamplified RNA/cDNA–Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses

                Infectious disease & Microbiology
                ebola virus,viruses,nanopore sequencing,venezuelan equine encephalitis virus,rna virus,genomics,sequencing,fieldable platform,zika virus

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