Milken Institute School of Public Health Poster Presentations (Marvin Center & Video)

Title

Designing a Targeted NGS Approach for Detecting Drug Resistant Mutations (DRMs) in HCV.

Poster Number

66

Document Type

Poster

Status

Graduate Student - Masters

Abstract Category

Epidemiology and Biostatistics

Keywords

Hepatitis C; Next-gen Sequencing; Drug resistant mutations

Publication Date

4-2017

Abstract

Hepatitis C virus (HCV) primarily affects the liver, if left untreated it can result in liver cancer or cirrhosis. HCV is a major public health concern and one of the few notifiable diseases in which mortality rate is still on the rise, especially in adults 50 years and older. A large percentage of infected individuals are unaware of their status. According to the Centers for Disease Control and Prevention there were over 30,500 new cases of HCV in the US in 2014, and an estimated 2.7-3.9 million individuals who are chronically infected.

Since 2011, treating HCV infections has become more effective with the introduction of a class of drugs called Direct Acting Antivirals (DAAs). DAAs target four specific nonstructural (NS) proteins: NS3/4A protease inhibitor (PIs), NS5A inhibitor, NS5B nucleoside polymerase inhibitor and non-nucleoside polymerase inhibitors. Currently the Food and Drug Administration has 10 approved DAAs for managing HCV. However, ~10-15% of individuals infected with HCV genotype 1, who have no history of exposure to NS5A inhibitors, are found to have detectable levels of HCV NS5A resistance-associated variants.

To address this concern and our desire to assess HCV DRMs, we designed HCV-specific PCR primer sets targeting 29 DRMs in 3 gene regions: NS3/4A (836 bp; 8 DRMs), NS5A (1032 bp; 6 DRMs) and NS5B (1460 bp; 15 DRMs). These primer sets recognize both HCV 1a and 1b subtypes, is the most prevalent genotype here in the US.

Ten de-identified HCV positive plasma samples (7; HCV 1a and 3; HCV 1b genotype) were used as controls in developing our targeted Next Generation Sequencing (NGS) approach. The viral load of these samples ranged from 3,067,577-12,502,372 copies/mL (mean; 5,823,201). After RNA extraction and cDNA synthesis, the 3 different HCV-specific primer sets were used to amplify NS3/4A, NS5A or NS5B regions. The sizes of the PCR amplicons were confirmed by gel electrophoresis, and concentrations determined using Qubit technology. DNA libraries were prepared using Nextera XT library preparation kit and the pooled libraries sequenced using MiSeq V2 chemistry (150x2). Data analysis on fastq files was carried out using CLCbio genomics workbench. Reference genomes were HCV 1a; EU256002 and HCV 1b; EU482883. Basic variant DRM analysis was completed for the 10 samples.

In conclusion, the results of this study illustrate our success in designing a targeted NGS approach for HCV sequencing across 3 regions encompassing 29 DRMs that will be useful tool for HCV molecular surveillance studies.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Open Access

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Poster to be presented at GW Annual Research Days 2017.

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Designing a Targeted NGS Approach for Detecting Drug Resistant Mutations (DRMs) in HCV.

Hepatitis C virus (HCV) primarily affects the liver, if left untreated it can result in liver cancer or cirrhosis. HCV is a major public health concern and one of the few notifiable diseases in which mortality rate is still on the rise, especially in adults 50 years and older. A large percentage of infected individuals are unaware of their status. According to the Centers for Disease Control and Prevention there were over 30,500 new cases of HCV in the US in 2014, and an estimated 2.7-3.9 million individuals who are chronically infected.

Since 2011, treating HCV infections has become more effective with the introduction of a class of drugs called Direct Acting Antivirals (DAAs). DAAs target four specific nonstructural (NS) proteins: NS3/4A protease inhibitor (PIs), NS5A inhibitor, NS5B nucleoside polymerase inhibitor and non-nucleoside polymerase inhibitors. Currently the Food and Drug Administration has 10 approved DAAs for managing HCV. However, ~10-15% of individuals infected with HCV genotype 1, who have no history of exposure to NS5A inhibitors, are found to have detectable levels of HCV NS5A resistance-associated variants.

To address this concern and our desire to assess HCV DRMs, we designed HCV-specific PCR primer sets targeting 29 DRMs in 3 gene regions: NS3/4A (836 bp; 8 DRMs), NS5A (1032 bp; 6 DRMs) and NS5B (1460 bp; 15 DRMs). These primer sets recognize both HCV 1a and 1b subtypes, is the most prevalent genotype here in the US.

Ten de-identified HCV positive plasma samples (7; HCV 1a and 3; HCV 1b genotype) were used as controls in developing our targeted Next Generation Sequencing (NGS) approach. The viral load of these samples ranged from 3,067,577-12,502,372 copies/mL (mean; 5,823,201). After RNA extraction and cDNA synthesis, the 3 different HCV-specific primer sets were used to amplify NS3/4A, NS5A or NS5B regions. The sizes of the PCR amplicons were confirmed by gel electrophoresis, and concentrations determined using Qubit technology. DNA libraries were prepared using Nextera XT library preparation kit and the pooled libraries sequenced using MiSeq V2 chemistry (150x2). Data analysis on fastq files was carried out using CLCbio genomics workbench. Reference genomes were HCV 1a; EU256002 and HCV 1b; EU482883. Basic variant DRM analysis was completed for the 10 samples.

In conclusion, the results of this study illustrate our success in designing a targeted NGS approach for HCV sequencing across 3 regions encompassing 29 DRMs that will be useful tool for HCV molecular surveillance studies.