Comparison of targeted next-generation sequencing for whole-genome sequencing of Hantaan orthohantavirus in Apodemus agrarius lung tissues

Sci Rep. 2019 Nov 12;9(1):16631. doi: 10.1038/s41598-019-53043-2.

Abstract

Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. In humans, orthohantavirus infection causes hemorrhagic fever with renal syndrome or hantavirus cardiopulmonary syndrome. Whole-genome sequencing of the virus helps in identification and characterization of emerging or re-emerging viruses. Next-generation sequencing (NGS) is a potent method to sequence the viral genome, using molecular enrichment methods, from clinical specimens containing low virus titers. Hence, a comparative study on the target enrichment NGS methods is required for whole-genome sequencing of orthohantavirus in clinical samples. In this study, we used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for whole-genome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. We analyzed the coverage of the HTNV genome based on the viral RNA copy number, which is quantified by real-time quantitative PCR. Target capture and amplicon NGS demonstrated a high coverage rate of HTNV in Apodemus agrarius lung tissues containing up to 103-104 copies/μL of HTNV RNA. Furthermore, the amplicon NGS showed a 10-fold (102 copies/μL) higher sensitivity than the target capture NGS. This report provides useful insights into target enrichment NGS for whole-genome sequencing of orthohantaviruses without cultivating the viruses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Genome, Viral / genetics
  • Hantaan virus / genetics*
  • High-Throughput Nucleotide Sequencing / methods*
  • Lung / virology*
  • Murinae / virology*
  • Phylogeny
  • Real-Time Polymerase Chain Reaction
  • Republic of Korea
  • Whole Genome Sequencing / methods*