A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus

Martin Schou Pedersen, Ulrik Fahnøe, Thomas Arn Hansen, Anders Gorm Pedersen, Håvard Jenssen, Jens Bukh, Kristian Schønning

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

BACKGROUND:
The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins.

OBJECTIVE:
To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype.

STUDY DESIGN:
In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs.

RESULTS:
The method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples.

CONCLUSIONS:
The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.
OriginalsprogEngelsk
TidsskriftJournal of Clinical Virology
Vol/bind105
Sider (fra-til)49-56
ISSN1386-6532
DOI
StatusUdgivet - 2018

Emneord

  • Genotyping
  • NGS
  • RAS
  • RT-PCR
  • Replicon
  • Subgenome

Citer dette

Pedersen, Martin Schou ; Fahnøe, Ulrik ; Hansen, Thomas Arn ; Pedersen, Anders Gorm ; Jenssen, Håvard ; Bukh, Jens ; Schønning, Kristian. / A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus. I: Journal of Clinical Virology. 2018 ; Bind 105. s. 49-56.
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title = "A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus",
abstract = "BACKGROUND:The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins.OBJECTIVE:To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype.STUDY DESIGN:In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs.RESULTS:The method successfully amplified and sequenced 94{\%} (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22{\%}) samples at a 15{\%} threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4{\%}) of 95 successfully sequenced samples.CONCLUSIONS:The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.",
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author = "Pedersen, {Martin Schou} and Ulrik Fahn{\o}e and Hansen, {Thomas Arn} and Pedersen, {Anders Gorm} and H{\aa}vard Jenssen and Jens Bukh and Kristian Sch{\o}nning",
year = "2018",
doi = "10.1016/j.jcv.2018.05.012",
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A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus. / Pedersen, Martin Schou; Fahnøe, Ulrik; Hansen, Thomas Arn; Pedersen, Anders Gorm; Jenssen, Håvard; Bukh, Jens; Schønning, Kristian.

I: Journal of Clinical Virology, Bind 105, 2018, s. 49-56.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - A near full-length open reading frame next generation sequencing assay for genotyping and identification of resistance-associated variants in hepatitis C virus

AU - Pedersen, Martin Schou

AU - Fahnøe, Ulrik

AU - Hansen, Thomas Arn

AU - Pedersen, Anders Gorm

AU - Jenssen, Håvard

AU - Bukh, Jens

AU - Schønning, Kristian

PY - 2018

Y1 - 2018

N2 - BACKGROUND:The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins.OBJECTIVE:To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype.STUDY DESIGN:In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs.RESULTS:The method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples.CONCLUSIONS:The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.

AB - BACKGROUND:The current treatment options for hepatitis C virus (HCV), based on direct acting antivirals (DAA), are dependent on virus genotype and previous treatment experience. Treatment failures have been associated with detection of resistance-associated substitutions (RASs) in the DAA targets of HCV, the NS3, NS5A and NS5 B proteins.OBJECTIVE:To develop a next generation sequencing based method that provides genotype and detection of HCV NS3, NS5A, and NS5 B RASs without prior knowledge of sample genotype.STUDY DESIGN:In total, 101 residual plasma samples from patients with HCV covering 10 different viral subtypes across 4 genotypes with viral loads of 3.84-7.61 Log IU/mL were included. All samples were de-identified and consequently prior treatment status for patients was unknown. Almost full open reading frame amplicons (∼ 9 kb) were generated using RT-PCR with a single primer set. The resulting amplicons were sequenced with high throughput sequencing and analysed using an in-house developed script for detecting RASs.RESULTS:The method successfully amplified and sequenced 94% (95/101) of samples with an average coverage of 14,035; four of six failed samples were genotype 4a. Samples analysed twice yielded reproducible nucleotide frequencies across all sites. RASs were detected in 21/95 (22%) samples at a 15% threshold. The method identified one patient infected with two genotype 2b variants, and the presence of subgenomic deletion variants in 8 (8.4%) of 95 successfully sequenced samples.CONCLUSIONS:The presented method may provide identification of HCV genotype, RASs detection, and detect multiple HCV infection without prior knowledge of sample genotype.

KW - Genotyping

KW - NGS

KW - RAS

KW - RT-PCR

KW - Replicon

KW - Subgenome

KW - Genotyping

KW - NGS

KW - RAS

KW - RT-PCR

KW - Replicon

KW - Subgenome

U2 - 10.1016/j.jcv.2018.05.012

DO - 10.1016/j.jcv.2018.05.012

M3 - Journal article

VL - 105

SP - 49

EP - 56

JO - Journal of Clinical Virology

JF - Journal of Clinical Virology

SN - 1386-6532

ER -