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      Arbovirus Detection in Insect Vectors by Rapid, High-Throughput Pyrosequencing

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          Despite the global threat caused by arthropod-borne viruses, there is not an efficient method for screening vector populations to detect novel viral sequences. Current viral detection and surveillance methods based on culture can be costly and time consuming and are predicated on prior knowledge of the etiologic agent, as they rely on specific oligonucleotide primers or antibodies. Therefore, these techniques may be unsuitable for situations when the causative agent of an outbreak is unknown.

          Methodology/Principal Findings

          In this study we explored the use of high-throughput pyrosequencing for surveillance of arthropod-borne RNA viruses. Dengue virus, a member of the positive strand RNA Flavivirus family that is transmitted by several members of the Aedes genus of mosquitoes, was used as a model. Aedes aegypti mosquitoes experimentally infected with dengue virus type 1 (DENV-1) were pooled with noninfected mosquitoes to simulate samples derived from ongoing arbovirus surveillance programs. Using random-primed methods, total RNA was reverse-transcribed and resulting cDNA subjected to 454 pyrosequencing.


          In two types of samples, one with 5 adult mosquitoes infected with DENV-1- and the other with 1 DENV-1 infected mosquito and 4 noninfected mosquitoes, we identified DENV-1 DNA sequences. DENV-1 sequences were not detected in an uninfected control pool of 5 adult mosquitoes. We calculated the proportion of the Ae. aegypti metagenome contributed by each infecting Dengue virus genome (p IP), which ranged from 2.75×10 −8 to 1.08×10 −7. DENV-1 RNA was sufficiently concentrated in the mosquito that its detection was feasible using current high-throughput sequencing instrumentation. We also identified some of the components of the mosquito microflora on the basis of the sequence of expressed RNA. This included members of the bacterial genera Pirellula and Asaia, various fungi, and a potentially uncharacterized mycovirus.

          Author Summary

          Traditional methods for virus detection often rely on specific attributes, such as DNA sequences, of the viruses and therefore they not only require a priori knowledge of the agent in question, but they also are generally very specific in nature, capable of detecting viruses only from within a specific family, for example. Nextgen sequencing shows much promise for detection/diagnostic applications because of its ever-increasing throughput, decreasing cost, and unbiased nature. We investigated the applicability of 454 pyrosequencing for viral surveillance of insect populations, using Aedes aegypti mosquitoes experimentally inoculated with Dengue virus type 1 (DENV-1) and calculated what proportion of the total nucleic acid from crushed mosquitoes was contributed by the virus. We concluded that 454 pyrosequencing is capable of detecting even very small amounts of a known virus from within a pool of infected and noninfected mosquitoes, but for the amount of sequencing reads required to detect the virus, this technique may currently be too cost-prohibitive for use in large-scale surveillance efforts. Interesting byproducts of our study included a glimpse into what symbiotic organisms Ae. aegypti may harbor, as well as what genes may be differentially expressed in a DENV-1-infected versus noninfected mosquito.

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          Most cited references 31

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          Basic local alignment search tool.

          A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.
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            Mapping and quantifying mammalian transcriptomes by RNA-Seq.

            We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41-52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3' untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 x 10(5) distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices.
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              Genome sequencing in microfabricated high-density picolitre reactors.

              The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

                Author and article information

                Role: Editor
                PLoS Negl Trop Dis
                PLoS Neglected Tropical Diseases
                Public Library of Science (San Francisco, USA )
                November 2010
                9 November 2010
                : 4
                : 11
                [1 ]Biological Defense Research Directorate, Naval Medical Research Center, Silver Spring, Maryland, United States of America
                [2 ]United States Army Medical Research Institute for Infectious Diseases, Fort Detrick, Maryland, United States of America
                Duke University-National University of Singapore, Singapore
                Author notes

                ¤: Current address: Department of Medicine, Division of Infectious Diseases and Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, United States of America

                Conceived and designed the experiments: KABL CAW TDR. Performed the experiments: KABL MJT KMW AB NMEN SML. Analyzed the data: KABL AA AM TNB SS CAW TDR. Wrote the paper: KABL SS TDR.

                This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
                Page count
                Pages: 10
                Research Article
                Genetics and Genomics/Gene Expression
                Virology/Effects of Virus Infection on Host Gene Expression
                Virology/Emerging Viral Diseases

                Infectious disease & Microbiology


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