A serotype-specific and tiled amplicon multiplex PCR method for whole genome sequencing of dengue virus

J Virol Methods. 2024 Jul:328:114968. doi: 10.1016/j.jviromet.2024.114968. Epub 2024 May 23.

Abstract

Dengue fever, a mosquito-borne viral disease of significant public health concern in tropical and subtropical regions, is caused by any of the four serotypes of the dengue virus (DENV1-4). Cutting-edge technologies like next-generation sequencing (NGS) are revolutionizing virology, enabling in-depth exploration of DENV's genetic diversity. Here, we present an optimized workflow for full-genome sequencing of DENV 1-4 utilizing tiled amplicon multiplex PCR and Illumina sequencing. Our assay, sequenced on the Illumina MiSeq platform, demonstrates its ability to recover the full-length dengue genome across various viral abundances in clinical specimens with high-quality base coverage. This high quality underscores its suitability for precise examination of intra-host diversity, enriching our understanding of viral evolution and holding potential for improved diagnostic and intervention strategies in regions facing dengue outbreaks.

Keywords: Dengue virus; amplicon multiplex PCR; low viral abundance; next-generation sequencing.

MeSH terms

  • Dengue Virus* / classification
  • Dengue Virus* / genetics
  • Dengue Virus* / isolation & purification
  • Dengue* / diagnosis
  • Dengue* / virology
  • Genome, Viral* / genetics
  • High-Throughput Nucleotide Sequencing* / methods
  • Humans
  • Multiplex Polymerase Chain Reaction* / methods
  • RNA, Viral / genetics
  • Serogroup*
  • Whole Genome Sequencing* / methods

Substances

  • RNA, Viral