Evaluation of high-throughput sequencing for identifying knwon and unknown viruses in biological samples
2011
Cheval, Justine | Sauvage, Virginie | Frangeul, Lionel | Dacheux, Laurent | Guignon, Ghislaine | Dumey, Nicolas | Pariente, Kevin | Rousseaux, Claudine | Dorange, Fabien | Berthet, Nicolas | Brisse, Sylvain | Moszer, Ivan | Bourhy, Hervé | Manuguerra, Jean-Claude | Lecuit, Marc | Burguiere, Ana | Caro, Valérie | Eloit, Marc | Génotypage des Pathogènes et Santé Publique (Plate-forme) (PF8) ; Institut Pasteur [Paris] (IP) | Cellule d'Intervention Biologique d'Urgence - Laboratory for Urgent Response to Biological Threats (CIBU) ; Institut Pasteur [Paris] (IP) | Intégration et Analyse Génomique (Plate-Forme 4) (PF4) ; Institut Pasteur [Paris] (IP) | Dynamique des Lyssavirus et Adaptation à l'Hôte (DyLAH) ; Institut Pasteur [Paris] (IP) | Texcell | Epidémiologie et Physiopathologie des Virus Oncogènes (EPVO (UMR_3569 / U-Pasteur_3)) ; Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS) | Microorganismes et Barrières de l'Hôte (Equipe avenir) ; Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM) | Département de Virologie - Department of Virology ; Institut Pasteur [Paris] (IP) | Virologie UMR1161 (VIRO) ; École nationale vétérinaire d'Alfort (ENVA)-Institut National de la Recherche Agronomique (INRA)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES) | The platform Genotyping of Pathogens and Public Health is supported in part by the Institut de Veille Sanitaire (Saint-Maurice, France). This study was mainly supported by Programme Transversal de Recherche (PATHODISC 301) from the Institut Pasteur (France) and by grants from region Ile de France.
法语. High-throughput sequencing furnishes a large number of short sequence reads from uncloned DNA and hasrapidly become a major tool for identifying viruses in biological samples, and in particular when the targetsequence is undefined. In this study, we assessed the analytical sensitivity of a pipeline for detection of virusesin biological samples based on either the Roche-454 genome sequencer or Illumina genome analyzer platforms.We sequenced biological samples artificially spiked with a wide range of viruses with genomes composed ofsingle or double-stranded DNA or RNA, including linear or circular single-stranded DNA. Viruses were addedat a very low concentration most often corresponding to 3 or 0.8 times the validated level of detection ofquantitative reverse transcriptase PCRs (RT-PCRs). For the viruses represented, or resembling those represented,in public nucleotide sequence databases, we show that the higher output of Illumina is associated witha much greater sensitivity, approaching that of optimized quantitative (RT-)PCRs. In this blind study,identification of viruses was achieved without incorrect identification. Nevertheless, at these low concentrations,the number of reads generated by the Illumina platform was too small to facilitate assembly of contigswithout the use of a reference sequence, thus precluding detection of unknown viruses. When the virus load wassufficiently high, de novo assembly permitted the generation of long contigs corresponding to nearly full-lengthgenomes and thus should facilitate the identification of novel viruses.
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