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

Jin Sun No,Michael R Wiley,Daesang Lee,Jin-Won Song,Seungchan Cho,Gustavo Palacios,Seong Tae Jeong,Seung-Ho Lee,Jeong-Ah Kim,Won-Keun Kim,Se Hun Gu,Dong Hyun Song

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.

Highlights

Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat
Laboratory diagnosis examined the presence of anti-Hantaan orthohantavirus (HTNV) immunoglobulin G (IgG) and HTNV RNA by immunofluorescence assay (IFA) and RT-polymerase chain reaction (PCR), respectively (Table 1, Supplementary Fig. 1)
RT-PCR, Nested Reverse Transcription-Polymerase Chain Reaction; Ct, Cycle threshold; Pos, Positive; IgG, Immunoglobulin G. *In dead rodents, samples for IFA test was obtained from the heart

Summary

Introduction

Orthohantaviruses, negative-sense single-strand tripartite RNA viruses, are a global public health threat. We used the sequence-independent, single-primer amplification, target capture, and amplicon NGS for wholegenome sequencing of Hantaan orthohantavirus (HTNV) from rodent specimens. To diagnose and detect extremely low-titer viruses, several NGS methods have been developed, including enrichment of viral nucleic acids by using target-specific oligonucleotide probes, removing host genome sequences, purifying virus-like particles, and performing small-RNA deep sequencing[13,14,15,16]. One of the most prominent techniques for random access to viral nucleic acids is sequence-independent, single-primer amplification (SISPA)[17,18] It was used for sequencing HTNV isolates collected from HFRS endemic areas[19]. In target capture NGS, the samples are subjected to fragmentation, random reverse-transcription, ligation with barcoded library adapters, and polymerase chain reaction (PCR) amplification These libraries are pooled and enriched using virus-specific probes. To evaluate three NGS methodologies, we analyzed and compared the depth of coverage and the recovery of virus genomes on a basis of the viral RNA copy number per μL

Methods

Results

Conclusion