Applied shotgun metagenomics approach for the genetic characterization of dengue viruses

Dengue virus (DENV), an arthropod-borne virus, has rapidly spread in recent years. DENV diagnosis is performed through virus serology, isolation or molecular detection, while genotyping is usually done through Sanger sequencing of the envelope gene. This study aimed to optimize the use of shotgun metagenomics and subsequent bioinformatics analysis to detect and type DENV directly from clinical samples without targeted amplification. Additionally, presence of DENV quasispecies (intra-host variation) was revealed by detecting single nucleotide variants. Viral RNA was isolated with or without DNase-I treatment from 17 DENV (1-4) positive blood samples. cDNA libraries were generated using either a combination of the NEBNext® RNA to synthesize cDNA followed by Nextera XT DNA library preparation, or the TruSeq RNA V2 (TS) library preparation kit. Libraries were sequenced using both the MiSeq and NextSeq. Bioinformatic analysis showed complete ORFs for all samples by all approaches, but longer contigs and higher sequencing depths were obtained with the TS kit. No differences were observed between MiSeq and NextSeq sequencing. Detection of multiple DENV serotypes in a single sample was feasible. Finally, results were obtained within three days with associated reagents costs between €130-170/sample. Therefore, shotgun metagenomics is suitable for identification and typing of DENV in a clinical setting.