Evaluation of high-throughput sequencing for identifying knwon and unknown viruses in biological samples

Justine Cheval (1) , Virginie Sauvage (2) , Lionel Frangeul (3) , Laurent Dacheux (4) , Ghislaine Guignon (1) , Nicolas Dumey (5) , Kevin Pariente (2) , Claudine Rousseaux (2) , Fabien Dorange (5) , Nicolas Berthet (6) , Sylvain Brisse (1) , Ivan Moszer (3) , Hervé Bourhy (4) , Jean-Claude Manuguerra (2) , Marc Lecuit (7) , Ana Burguiere (2) , Valérie Caro (1) , Marc Eloit (8, 9)

1 PF8 - Génotypage des Pathogènes et Santé Publique (Plate-forme)
2 CIBU - Cellule d'Intervention Biologique d'Urgence - Laboratory for Urgent Response to Biological Threats
3 PF4 - Intégration et Analyse Génomique (Plate-Forme 4)
4 DyLAH - Dynamique des Lyssavirus et Adaptation à l'Hôte
5 Texcell
6 EPVO (UMR_3569 / U-Pasteur_3) - Epidémiologie et Physiopathologie des Virus Oncogènes
7 Equipe avenir - Microorganismes et Barrières de l'Hôte
8 Département de Virologie - Department of Virology
9 VIRO - Virologie UMR1161

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.

Mots clés: viruses; DNA; Identifying; Sequencing

Domaines: Sciences du Vivant [q-bio]

Justine Cheval, Virginie Sauvage, Lionel Frangeul, Laurent Dacheux, Ghislaine Guignon, et al.. Evaluation of high-throughput sequencing for identifying knwon and unknown viruses in biological samples. Journal of Clinical Microbiology, 2011, 49 (9), pp.3268-3276. ⟨10.1128/JCM.00850-11⟩. ⟨hal-01191107⟩