1. Utility of a Sequence-Independent, Single-Primer-Amplification (SISPA) and Nanopore Sequencing Approach for Detection and Characterization of Tick-Borne Viral Pathogens.
- Author
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Brinkmann A, Uddin S, Krause E, Surtees R, Dinçer E, Kar S, Hacıoğlu S, Özkul A, Ergünay K, and Nitsche A
- Subjects
- Animals, Arboviruses genetics, Flaviviridae genetics, Flaviviridae isolation & purification, Genome, Viral genetics, Hemorrhagic Fever Virus, Crimean-Congo genetics, Hemorrhagic Fever Virus, Crimean-Congo isolation & purification, High-Throughput Nucleotide Sequencing, Phylogeny, Arboviruses isolation & purification, DNA Primers genetics, Nanopore Sequencing methods, Ticks virology
- Abstract
Currently, next generation sequencing (NGS) is the mainly used approach for identification and monitorization of viruses with a potential public health threat in clinical and environmental samples. To facilitate detection in NGS, the sequence-independent, single-primer-amplification (SISPA) is an effective tool for enriching virus sequences. We performed a preliminary assessment of SISPA-nanopore sequencing as a potential approach for screening tick-borne viruses in six specimens with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen tick virus (JMTV) sequences. A comparison of unbiased NGS and SISPA followed by nanopore sequencing was carried out in 4 specimens with single and pooled ticks. The approach was further used for genome sequencing in culture-grown viruses. Overall, total/virus-specific read counts were significantly elevated in cell culture supernatants in comparison to single or pooled ticks. Virus genomes could be successfully characterized by SISPA with identities over 99%. Genome coverage varied according to the segment and total read count. Base calling errors were mainly observed in tick specimens and more frequent in lower viral loads. Culture-grown viruses were phylogenetically-related to previously-reported local viruses. In conclusion, the SISPA + nanopore sequencing was successful in generating data comparable to NGS and will provide an effective tool for broad-range virus detection in ticks.
- Published
- 2021
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