Voprosy virusologii · 2025
NGS amplification panel HCV-seq for sequencing hepatitis C virus RNA (Flaviviridae: Hepacivirus).
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Chanyshev M D, Chernyshova A S, Glushchenko A G, Grishaeva A A, Makashova V V, Ponezheva Z B, Khafizov K F, Akimkin V G
Аннотация
INTRODUCTION: Hepatitis C is a pressing global public health issue. The high variability of the hepatitis C virus (HCV) complicates its whole-genome sequencing; most studies sequence only specific regions of the genome. There is a need for a simple and reliable method for sequencing the whole genome of HCV.
OBJECTIVE: Development and validation of NGS panel for whole-genome sequencing of HCV.
MATERIALS AND METHODS: This study presents NGS amplification panel for sequencing the genomes of HCV genotypes 1, 2, and 3. Depending on the genotype, a set of 79, 67, or 89 primers is used. These primers enable amplification of overlapping regions of the HCV genome.
RESULTS: The panel was tested on 153 HCV RNA samples isolated from blood plasma specimens (93/6/54 samples of genotypes 1/2/3, respectively). Shannon entropy analysis showed that genetic heterogeneity within the E2 gene is significantly higher than in other parts of viral genome. The frequency of mutations associated with drug resistance was determined. Specifically, for genotype 1, the following mutation detection rates were observed in NS3: Y56F - 37.6%, V170I - 23.7%; in NS5a: R30Q - 8.6%, P58L/S/T - 6.5%, A92T - 4.3%; in NS5b: L159F - 45.2%, S556G/N - 33.3%.
CONCLUSION: The current study describes a method for whole-genome sequencing of HCV genotypes 1, 2, and 3. The HCV sequencing panel shows great potential for use in scientific research and epidemiological monitoring.
OBJECTIVE: Development and validation of NGS panel for whole-genome sequencing of HCV.
MATERIALS AND METHODS: This study presents NGS amplification panel for sequencing the genomes of HCV genotypes 1, 2, and 3. Depending on the genotype, a set of 79, 67, or 89 primers is used. These primers enable amplification of overlapping regions of the HCV genome.
RESULTS: The panel was tested on 153 HCV RNA samples isolated from blood plasma specimens (93/6/54 samples of genotypes 1/2/3, respectively). Shannon entropy analysis showed that genetic heterogeneity within the E2 gene is significantly higher than in other parts of viral genome. The frequency of mutations associated with drug resistance was determined. Specifically, for genotype 1, the following mutation detection rates were observed in NS3: Y56F - 37.6%, V170I - 23.7%; in NS5a: R30Q - 8.6%, P58L/S/T - 6.5%, A92T - 4.3%; in NS5b: L159F - 45.2%, S556G/N - 33.3%.
CONCLUSION: The current study describes a method for whole-genome sequencing of HCV genotypes 1, 2, and 3. The HCV sequencing panel shows great potential for use in scientific research and epidemiological monitoring.