comparison of shotgun metagenomic and targeted amplicon-based sequencing
Samo Zakotnik (Author), Nataša Knap (Author), Petra Bogovič (Author), Tomaž Mark Zorec (Author), Mario Poljak (Author), Franc Strle (Author), Tatjana Avšič-Županc (Author), Miša Korva (Author)

Abstract

The clinical presentation of tick-borne encephalitis virus (TBEV) infection varies from asymptomatic to severe meningoencephalitis or meningoencephalomyelitis. The TBEV subtype has been suggested as one of the most important risk factors for disease severity, but TBEV genetic characterization is difficult. Infection is usually diagnosed in the post-viremic phase, and so relevant clinical samples of TBEV are extremely rare and, when present, are associated with low viral loads. To date, only two complete TBEV genomes sequenced directly from patient clinical samples are publicly available. The aim of this study was to develop novel protocols for the direct sequencing of the TBEV genome, enabling studies of viral genetic determinants that influence disease severity. We developed a novel oligonucleotide primer scheme for amplification of the complete TBEV genome. The primer set was tested on 21 clinical samples with various viral loads and collected over a 15-year period using the two most common sequencing platforms. The amplicon-based strategy was compared to direct shotgun sequencing. Using the novel primer set, we successfully obtained nearly complete TBEV genomes (>90% of genome) from all clinical samples, including those with extremely low viral loads. Comparison of consensus sequences of the TBEV genome generated using the novel amplicon-based strategy and shotgun sequencing showed no difference. We conclude that the novel primer set is a powerful tool for future studies on genetic determinants of TBEV that influence disease severity and will lead to a better understanding of TBE pathogenesis.

Keywords

tick-borne encephalitis virus;whole genome sequencing;metagenomics;

Data

Language: English
Year of publishing:
Typology: 1.01 - Original Scientific Article
Organization: UL MF - Faculty of Medicine
UDC: 616.9
COBISS: 111682563 Link will open in a new window
ISSN: 1999-4915
Views: 16
Downloads: 4
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Other data

Secondary language: Slovenian
Secondary keywords: virus klopnega encefalitisa;sekvenciranje celotnega genoma;metagenomika;
Type (COBISS): Article
Pages: str. 1-16
Volume: ǂVol. ǂ14
Issue: ǂiss. ǂ6
Chronology: 2022
DOI: 10.3390/v14061267
ID: 18583948