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Detection of Natural Resistance-Associated Substitutions by Ion Semiconductor Technology in HCV1b Positive, Direct-Acting Antiviral Agents-Na ï ve Patients

View Article: PubMed Central - PubMed

ABSTRACT

Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-naïve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure.

No MeSH data available.


Workflow of sample-specific consensus sequence of the NS3 region generated by an in-house-developed script.
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ijms-17-01416-f003: Workflow of sample-specific consensus sequence of the NS3 region generated by an in-house-developed script.

Mentions: We provide an overview of the sequence read post-processing steps in Figure 3. Briefly, Cutadapt [31] and Sickle tools [30] were used to remove low quality reads, low complexity reads, adapters and primers from fastq raw sequence data. After quality control of raw data using FastQC (Available online: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and pre-processing with the Pollux error correction software [32], a de novo assembly was performed using software packages VICUNA [33] and V-FAT [34]. The sample-specific majority rule consensus sequences were used to infer the variant frequencies with V-phaser 2 [50], and we only considered variants that had no strand bias (p-value ≤ 0.05).


Detection of Natural Resistance-Associated Substitutions by Ion Semiconductor Technology in HCV1b Positive, Direct-Acting Antiviral Agents-Na ï ve Patients
Workflow of sample-specific consensus sequence of the NS3 region generated by an in-house-developed script.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037695&req=5

ijms-17-01416-f003: Workflow of sample-specific consensus sequence of the NS3 region generated by an in-house-developed script.
Mentions: We provide an overview of the sequence read post-processing steps in Figure 3. Briefly, Cutadapt [31] and Sickle tools [30] were used to remove low quality reads, low complexity reads, adapters and primers from fastq raw sequence data. After quality control of raw data using FastQC (Available online: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and pre-processing with the Pollux error correction software [32], a de novo assembly was performed using software packages VICUNA [33] and V-FAT [34]. The sample-specific majority rule consensus sequences were used to infer the variant frequencies with V-phaser 2 [50], and we only considered variants that had no strand bias (p-value ≤ 0.05).

View Article: PubMed Central - PubMed

ABSTRACT

Naturally occurring resistance-associated substitutions (RASs) can negatively impact the response to direct-acting antivirals (DAAs) agents-based therapies for hepatitis C virus (HCV) infection. Herein, we set out to characterize the RASs in the HCV1b genome from serum samples of DAA-naïve patients in the context of the SINERGIE (South Italian Network for Rational Guidelines and International Epidemiology, 2014) project. We deep-sequenced the NS3/4A protease region of the viral population using the Ion Torrent Personal Genome Machine, and patient-specific majority rule consensus sequence summaries were constructed with a combination of freely available next generation sequencing data analysis software. We detected NS3/4A protease major and minor variants associated with resistance to boceprevir (V36L), telaprevir (V36L, I132V), simeprevir (V36L), and grazoprevir (V36L, V170I). Furthermore, we sequenced part of HCV NS5B polymerase using Sanger-sequencing and detected a natural RAS for dasabuvir (C316N). This mutation could be important for treatment strategies in cases of previous therapy failure.

No MeSH data available.