Abstract
Persons with hepatitis C virus (HCV) genotype 1a (GT1a) infections harboring a baseline Q80K polymorphism in nonstructural protein 3 (NS3) have a reduced virologic response to simeprevir in combination with pegylated interferon-alfa and ribavirin. We aimed to develop, validate, and freely disseminate an NS3 clinical sequencing assay to detect the Q80K polymorphism and potentially other HCV NS3 drug resistance mutations. HCV RNA was extracted from frozen plasma using a NucliSENS easyMAG automated nucleic acid extractor, amplified by nested reverse transcription-PCR, and sequenced using Sanger and/or next-generation (MiSeq) methods. Sanger chromatograms were analyzed using in-house software (RECall), and nucleotide mixtures were called automatically. MiSeq reads were iteratively mapped to the H77 reference genome, and consensus NS3 sequences were generated with nucleotides present at >20% called as mixtures. The accuracy, precision, and sensitivity for detecting the Q80K polymorphism were assessed in 70 samples previously sequenced by an external laboratory. A comparison of the sequences generated by the Sanger and MiSeq methods with those determined by an external lab revealed >98.5% nucleotide sequence concordance and zero discordant calls of the Q80K polymorphism. The results were both highly repeatable and reproducible (>99.7% nucleotide concordance and 100% Q80K concordance). The limits of detection (>2 and ∼5 log10 IU/ml for the Sanger and MiSeq assays, respectively) are sufficiently low to allow genotyping in nearly all chronically infected treatment-naive persons. No systematic bias in the under- or overamplification of minority variants was observed. Coinfection with other viruses (e.g., HIV and hepatitis B virus [HBV]) did not affect the assay results. The two independent HCV NS3 sequencing assays with the automated analysis procedures described here are useful tools to screen for the Q80K polymorphism and other HCV protease inhibitor drug resistance mutations.
INTRODUCTION
Until recently, the standard of care for treating hepatitis C virus (HCV) infection has been combination antiviral therapy with pegylated interferon-alfa and ribavirin (Peg-IFN/RBV). In 2011, the nonstructural protein 3 (NS3) protease inhibitors (PI) telaprevir and boceprevir, in combination with Peg-IFN/RBV, were the first direct-acting antiviral (DAA) agents approved for treatment of chronic HCV genotype 1 infection (1–3). The success of HCV therapy with DAA, however, is complicated by the incredible genetic diversity of the virus and its capacity to mutate in response to drug selection pressure (4, 5). Treatment failure is often accompanied by the emergence of resistance mutations in the genes targeted by these drugs (6, 7). Furthermore, certain drug resistance mutations exist as naturally occurring polymorphisms in a small proportion of treatment-naive patients and can compromise PI treatment in these individuals (8–11).
In combination with Peg-IFN/RBV, the second-generation PI simeprevir was approved in Canada in 2013 for the treatment of chronic HCV genotype 1 infection in adults. The simeprevir combination was shown to be superior to Peg-IFN/RBV alone, with a sustained virologic response (SVR) of >80% being achieved in both the QUEST-1 and QUEST-2 phase III clinical trials (12, 13). However, the SVR rates for the simeprevir combination were reduced to 58% in patients having HCV genotype 1a (GT1a) infection with the NS3 Q80K polymorphism at baseline; this SVR rate was nonsuperior to that observed in the placebo arm. Overall, 56% of the patients with GT1a infection who did not achieve an SVR in the simeprevir arms had the NS3 Q80K polymorphism at baseline. In subsequent retrospective genotyping studies, it was discovered that approximately 30% of the patients with GT1a infection who enrolled in the phase II and III clinical trials of simeprevir had HCV harboring the Q80K polymorphism at baseline (14). In addition, a significant geographic bias in the distribution of the Q80K polymorphism was discovered: 48% of patients with HCV GT1a in North America had the Q80K polymorphism at baseline, compared to 19% of patients in Europe. In contrast, only 0.5% of patients with HCV GT1b were infected with viruses carrying the Q80K polymorphism, and no geographical differences were observed. The Q80K polymorphism is stable; viruses carrying the polymorphism are transmissible and are likely descended from a single lineage originating in the United States, in which the Q80K substitution arose around the 1940s (15).
Owing to the stability and high frequency of this polymorphism in Europe and especially in North America, screening for the Q80K polymorphism is strongly recommended before initiating simeprevir, pegylated interferon, and ribavirin combination therapy in patients with HCV GT1a infection (16). Here, we present the methods and demonstrate the performance of two independent HCV NS3 Q80K polymorphism assays involving nested–RT-PCR and sequencing of a portion of the NS3 protease region: (i) a Sanger sequencing approach incorporating primary and secondary PCR methods, and (ii) a next-generation sequencing approach involving near-whole-genome amplification and sequencing on an Illumina MiSeq.
MATERIALS AND METHODS
Samples.
Janssen Diagnostics BVBA provided frozen plasma samples from 70 treatment-naive HCV genotype 1-infected participants from the QUEST-1 and QUEST-2 phase III clinical trials of simeprevir in order to test sequencing accuracy. The median HCV plasma viral load (pVL) was 6.7 log10 IU/ml (interquartile range [IQR], 6.1 to 6.9 log10 IU/ml; range, 4.9 to 7.5 log10 IU/ml). HCV NS3 was previously sequenced in these samples at the Janssen Diagnostics Laboratory in Beerse, Belgium. The BC Centre for Excellence in HIV/AIDS (BCCfE) laboratory remained blinded to the Janssen sequencing results and sample collection details (study arm and timing) throughout assay development and validation.
In addition, archived frozen (−70°C) plasma samples from HCV genotype 1-infected participants of the Vancouver Injection Drug Users Study (VIDUS) were screened to identify two sample groups, one having wild-type virus, and one with the Q80K polymorphism. These sample sets were used for inter- and intra-assay precision studies. The HCV pVL was unknown for these samples.
A single HCV-positive GT1a plasma sample with a pVL of 6.6 log10 IU/ml (SeraCare Life Sciences, Milford, MA, USA) was spiked with HIV-positive plasma and HBV-positive plasma to test potential interference by other viruses. An HCV subtype panel (SeraCare) was used to test cross-reactivity across HCV genotypes (pVL range, 3.7 to 4.2 log10 IU/ml). Finally, in order to investigate analytical specificity, HCV-negative samples were tested: diethyl pyrocarbonate (DEPC)-treated water, pooled normal human plasma, HBV-positive/HCV-negative plasma (SeraCare), and in-house HIV-positive/HCV-negative controls.
Ethical approval was granted by the University of British Columbia Providence Health Care Research Ethics Board (H13-03520).
RNA extraction.
HCV viral RNA was extracted from 500 μl of frozen plasma using a NucliSENS easyMAG automated nucleic acid extractor (bioMérieux Canada, Saint-Laurent, Quebec, Canada), as per the manufacturer's instructions. Extracted RNA was eluted in 60 μl of elution buffer and stored at −20°C until RT-PCR amplification. When 60 μl of extracted RNA was insufficient for the intended experiments, multiple extractions were performed and the eluates pooled before further processing.
One-step RT-PCR.
Extracted HCV RNA was amplified using the Qiagen OneStep RT-PCR kit (Qiagen Sciences, Valencia, CA, USA), followed by an in-house second-round nested-PCR protocol. Two independent nested–RT-PCR amplification reactions intended to be primary and secondary (to be used in case of initial assay failure) methods were designed and tested in parallel. Both amplicons were designed to cover the Q80 polymorphism and all the major NS3 protease inhibitor resistance mutations V36A/M, T54A/S, V55A, S122R, R155K/Q, A156S/G, D168A/E/H/T/V/Y, and V/I170A. As simeprevir is approved only for the treatment of HCV genotype 1 infections and the Q80K polymorphism is mainly limited to HCV GT1a, the assay primers were designed to maximize the amplification success rate of HCV GT1 samples.
The primary ∼1.4-kb amplicon was generated using the reverse transcription primer NSR1 and two forward first-round PCR primers, NSF1 and 5HCPROT1 (see Table S1 in the supplemental material for PCR primer sequences). Briefly, 8 μl of extracted RNA was used to generate and amplify cDNA into a total reaction volume of 40 μl, consisting of 17.35 μl of DEPC-treated water, 8 μl of 5× OneStep RT-PCR buffer (Qiagen), 1.6 μl of 10 mM deoxynucleoside triphosphate (dNTP) mix (Qiagen), 1.2 μl of 25 μM NSR1 primer, 1 μl of 25 μM NSF1 primer, 1 μl of 25 μM 5HCPROT1 primer, 0.25 μl of Protector RNase inhibitor (Roche), and 1.6 μl of OneStep RT-PCR enzyme mix (Qiagen). The thermal cycling conditions were 54°C for 30 min, 95°C for 15 min, 8 cycles of 94°C for 30 s, 64°C for 30 s (−0.5°C/cycle), and 72°C for 80 s, and 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 85 s.
The secondary ∼800-bp amplicon was generated using reverse transcription primer HCV1NS3SR1 and forward first-round PCR primer HCV1NS3SF1. The thermal cycling conditions and reaction mixes were modified slightly from those of the primary method. Briefly, 1.2 μl each of 25 μM HCV1NS3SR1 and 25 μM HCV1NS3SF1 primer were used, and an additional 0.8 μl of DEPC-treated water was used to bring the total reaction mixture volume to 40 μl. The RT-PCR thermal cycling conditions for the secondary method were 54°C for 30 min, 95°C for 15 min, 10 cycles of 94°C for 30 s, 62°C for 30 s (−0.5°C/cycle), and 72°C for 45 s, and 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 50 s.
Amplification of HCV NS3 by nested-PCR and Sanger sequencing.
For the primary method, a second nested-PCR using forward primer NSF2 and reverse primers NSR2 and 3HCPROT2 was performed. The total reaction volume of 20 μl consisted of 2 μl of first-round PCR product, 12.39 μl of DEPC-treated water, 2 μl of 60% sucrose with 0.08% cresol red, 2 μl of Expand High Fidelity 10× buffer with 15 mM MgCl2 (Roche), 0.8 μl of 25 mM MgCl2 stock solution (Roche), 0.16 μl of 100 mM dNTP (Roche), 0.29 μl of Expand High Fidelity enzyme mix (Roche), and 0.12 μl each of 25 μM primers NSF2, NSR2, and 3HCPROT2. The thermal cycling conditions for the second-round nested-PCR were 95°C for 3 min, 8 cycles of 94°C for 15 s, 64°C for 30 s (−0.5°C/cycle), and 72°C for 75 s, and 30 cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 75 s (+3 s/cycle).
The secondary PCR method used the second-round nested-PCR forward primer HCV1NS3SF2 and reverse primer HCV1NS3SR2. PCR primers from the primary nested-PCR mix were substituted with 0.12 μl each of 25 μM primers HCV1NS3SF2 and HCV1NS3SR2 and DEPC-treated water. The thermal cycling conditions for the secondary method were 95°C for 3 min, 10 cycles of 94°C for 15 s, 64°C for 30 s (−0.5°C/cycle), and 72°C for 45 s, and 30 cycles of 94°C for 15 s, 58°C for 30 s, and 72°C for 50 s (+3 s/cycle).
Bulk (population) sequencing was performed on the amplified products on an ABI 3730xl DNA analyzer (Life Technologies, Carlsbad, CA, USA). The sequencing primers are listed in Table S2 in the supplemental material. Chromatograms were analyzed by the in-house software RECall (version 2.25) (17). To account for the substantial variation between genotypes, sequence reads were aligned against a set of seven HCV NS3 genotype consensus sequences (1a, 1b, 2a, 2b, 3a, 4, and 6a), with the highest-scoring reference being subsequently chosen for contig assembly. Nucleotide mixtures were called automatically when the secondary peak area was >20% of the major peak. No human editing of base calls was performed. Software settings allowed single primer coverage except at NS3 codon 80, and short sections of poor-quality sequence in regions not affecting Q80K calls may have been excluded. The final assembled sequences covered 1,200 bp (H77 positions 3420 to 4619) and 564 bp (H77 positions 3420 to 3983) for the primary and secondary assays, respectively.
Illumina MiSeq sample preparation and sequencing.
In addition to the NS3 amplicon protocol using Sanger sequencing, a separate assay using next-generation (deep) sequencing of the whole HCV genome was also investigated as an independent comparison. Briefly, a near-full-length amplicon was generated, according to a previously published protocol (18), using an oligo(dA)20 primer for cDNA synthesis. Two nested-PCRs using primers optimized for genotypes 1a and 1b were used to generate an 8,991-bp amplicon spanning the core to partial NS5B proteins. Following second-round PCR amplification, libraries for MiSeq sequencing were prepared using Nextera XT DNA sample preparation kits (Illumina, San Diego, CA, USA), according to the manufacturer's specifications. Amplicons were uniquely tagged using a dual-indexing approach (Nextera XT index kit; Illumina), multiplexed 48-fold, and sequenced using MiSeq version 2 paired-end kits (2 × 250bp).
The resulting reads were processed using an in-house pipeline that incorporated iterative short-read mapping with Bowtie 2 (19) and SAMtools (20). Briefly, paired-end reads were initially mapped to the H77 reference genome using default settings for local alignment (19). Mapped reads were collapsed into a sample-specific consensus to which all sequenced reads were subsequently remapped. Consensus building and remapping proceeded in an iterative fashion until ≥95% of the reads mapped or no additional reads could be recovered by additional rounds of remapping. The overlapping portions of paired-end reads were merged, and error correction rules were applied: bases with sequencing quality scores of <15 were discarded, and conflicting base calls in overlapping reads were resolved by retaining the base with the higher quality score. In order to compare the MiSeq and Sanger sequencing results, consensus sequences for NS3 were generated from the resulting SAM files, with nucleotides with >20% frequency being called as mixtures to mimic the expected sensitivity of Sanger sequencing. Samples with <1,000-fold coverage at NS3 codon 80 were excluded from downstream analysis.
Assessment of performance characteristics.
Three metrics were used to assess the quality of PCR amplification and sequencing between methods and/or replicates: concordance of nucleotide base calls, concordance of amino acid sequences, and concordance of the presence or absence of the NS3 Q80K polymorphism. Concordance was calculated as the proportion of nucleotide or amino acid agreement observed across all the nucleotide/amino acids sequenced. For the purpose of validation, partial discordances in nucleotide or amino acid calls (when one method observed a mixture while the other method detected only one component thereof, e.g., nucleotides Y versus C) were weighted equally as complete discordances. In analyses involving amplification and sequencing of multiple replicates of a sample for which no gold standard reference sequence was available, a consensus sequence constructed from all available replicates was used as the comparator. Nucleotides appearing in ≥20% of the replicate sequences were included (as mixtures) in the consensus.
Assay accuracy.
The sequences obtained from 70 plasma samples were compared to those previously generated by an independent laboratory (Janssen Diagnostics), who performed the testing for the QUEST clinical trials.
Assay precision.
Repeatability (intra-assay variability) was tested in three samples: one commercially prepared HCV-positive plasma and two clinically derived samples (one with Q80K/R and one without). Twelve replicates of each sample were PCR amplified and sequenced in a single batch. Reproducibility (interassay variability) was tested in 11 samples from the VIDUS cohort, with four samples with the Q80K/R polymorphism, and seven with wild-type Q80Q. All samples were tested on five different days by two laboratory technicians.
Assay sensitivity.
Assay sensitivity, defined as the lowest HCV RNA concentration that could be amplified in a minimum of two-thirds of the samples attempted, was determined using five patient samples from the Janssen Diagnostics sample set. Samples with an HCV pVL between 5.6 and 6.0 log10 IU/ml were serially diluted (1:10) with pooled normal human plasma to obtain concentrations in the ∼2-log10 IU/ml range. HCV RNA was extracted from diluted plasma samples, and six replicates of three samples per method were tested in a single batch.
Assay specificity.
Specificity was assessed via three experiments. First, five HCV-negative samples (pooled normal human plasma, HBV-positive/HCV-negative plasma, two in-house HIV-positive/HCV-negative controls, and DEPC-treated water) were extracted, amplified in triplicate, and gel electrophoresis was run on the amplified products. Second, PCR cross-reactivity or interference by other viruses was evaluated in two HCV-positive samples spiked with either HIV-1 or HBV. HCV-positive plasma with a pVL of 6.6 log10 IU/ml was spiked with a clinically derived HIV-positive plasma sample (pVL, 3.9 log10 HIV RNA copies/ml; primary method), an HIV laboratory clone (PNL4-3; pVL, approximately 3.9 log10 HIV RNA copies/ml; secondary method), or HBV-positive plasma (primary and secondary methods). The HBV- and HIV-spiked plasma samples were amplified and sequenced in triplicate in a single batch. Finally, the ability to amplify products from non-GT1 was evaluated using a commercial panel of HCV subtypes (SeraCare). The panel included samples designated genotypes 1a, 1b, 2a/c, 2b, 3a, 4, 4a/c/d, 5a, and 6a/b and one HCV-negative plasma sample. The panel samples were amplified in triplicate in a single batch.
All validation experiments were performed using both the primary and secondary Sanger sequencing methods. Validation of the MiSeq method was limited to the accuracy, reproducibility, and sensitivity experiments.
RESULTS
In this study, we developed and characterized two HCV NS3 sequencing assays intended to screen for the Q80K polymorphism prior to the initiation of simeprevir-containing therapy. The Sanger sequencing assay makes use of two independent PCR amplifications: a primary method that produces a 1,200-bp sequence, and a secondary method that produces a 564-bp sequence to be used as a backup in the event that amplification with the primary primers fails. For the purpose of this validation, primary and secondary methods were considered independent tests, and all validation experiments were performed using both methods. In addition, we developed an independent next-generation sequencing assay involving near-full-genome amplification of HCV genotype 1, followed by Nextera XT library preparation (Illumina).
Assay accuracy. (i) Concordance of Sanger sequences with external laboratory results.
Janssen Diagnostics provided 70 frozen plasma samples from treatment-naive HCV GT1-infected participants of the simeprevir licensing trials (86% GT1a and 14% GT1b). The samples had a median pVL of 6.7 log10 IU/ml (IQR, 6.1 to 6.9 log10 IU/ml). Nucleotide sequences determined by Janssen (either a 2,055-bp sequence covering the entire NS3 and NS4a genes, or a 543-bp fragment covering the first 181 codons of NS3) served as the comparator. According to the Janssen assay, 30 of 70 (42.9%) samples contained the Q80K polymorphism alone or as part of a mixture. Using the primary and secondary methods described here, all 30 Q80K polymorphisms were identified in HCV GT1a samples.
Amplification and sequencing by the primary 1,200-bp assay was successful in 66 (94.3%) samples. Compared to the Janssen sequences, we observed 98.8%, 99.6%, and 100% overall concordance in nucleotide sequences, amino acid sequences, and Q80K calls, respectively (see Fig. S1A in the supplemental material). When sequences were compared individually, a median 99.3% (IQR, 98.2 to 99.8%) nucleotide concordance was observed between sequence pairs. The vast majority of discrepancies (97.8% of nucleotide and 98.9% of amino acid differences) were due to differences in mixture calls (Fig. 1), with the BCCfE primary method calling a marginally higher number of mixed bases overall; a total of 1.5% and 1.3% of all bases were called as mixtures by the BCCfE primary and Janssen methods, respectively. Despite the small difference in the number of mixed bases called, we observed no systematic bias toward over- or undercalling mixtures by either the BCCfE primary or Janssen assay (Fig. 2).
FIG 1.
Summary of nucleotide sequence discordances between HCV NS3 sequencing assays developed by BCCfE and results obtained by Janssen Diagnostics. Three versions of the BCCfE assay were investigated: a primary Sanger sequencing method, which produces a 1,200-bp sequence; a secondary Sanger sequencing method, which produces a 564-bp sequence; and a next-generation sequencing assay using the Illumina MiSeq. Sequences obtained by BCCfE methods were compared to the Sanger sequences obtained by Janssen. All assays achieved ≥98.8% concordance in nucleotide base calls. Nearly all discordant base calls (>97.5% for Sanger and 91.7% for MiSeq) were due to differences in mixture calling between laboratories (blue bars), rather than completely incompatible base calls (orange bars). All instances of the Q80K polymorphism (n = 30) detected by the Janssen method were also detected by all three BCCfE methods.
FIG 2.
Analysis of the number of mixed bases per sequence suggests no systematic bias in amplification of minority species. The percentage of ambiguous nucleotides (mixed bases) in the longest fragment sequenced by both the BCCfE and Janssen laboratories (543, 564, or 1,200 bp) was compared. A modest, but not perfect, correlation (A), consistent with variation in RNA extraction, RT-PCR amplification, and base calling, was observed between sequences collected in the two laboratories. However, inspection of the Bland-Altman plot (B) revealed no systematic over- or undercalling of mixtures by either laboratory.
In total, 68 (97.1%) samples were successfully amplified and sequenced using the BCCfE secondary method. Compared to the sequences provided by Janssen, we observed 98.8%, 99.5%, and 100% concordance in nucleotide sequences, amino acid sequences, and Q80K calls, respectively (see Fig. S1B in the supplemental material). The median nucleotide concordance between sequence pairs was 99.1% (IQR, 97.8 to 100%). As in the primary assay, almost all sequence discrepancies were due to differences in mixture calls (98.5% of nucleotide and 100% of amino acid differences) (Fig. 1). In one sample, the BCCfE secondary method identified a Q80Q/K mixture, whereas the Janssen method observed a Q80K polymorphism; however, this partial amino acid difference would have no impact on a resistance interpretation, as both methods identified the Q80K polymorphism.
For completeness, the sequencing results from the primary and secondary amplification methods were compared to each other. In total, 64 (91.4%) samples gave results by both methods. Overall, we observed 99.0%, 99.7%, and 100% concordance in nucleotide sequences, amino acid sequences, and Q80K calls, respectively (data not shown). As the two Sanger sequencing methods are intended to serve as primary and secondary (backup) methods in a single resistance testing protocol, it should be noted that all 70 samples were successfully sequenced by at least one of the two methods.
(ii) MiSeq results.
Near-full-genome HCV was amplified from frozen plasma samples provided by Janssen Diagnostics. After excluding samples with <1,000-fold coverage at NS3 codon 80 following demultiplexing, quality control, and iterative mapping, consensus NS3 sequences were successfully obtained for 67 (96%) samples. The median coverage at codon 80 was 8,800 reads/sample (IQR, 6,500 to 11,000 reads/sample) and was fairly consistent across the length of NS3. While not the subject of the current study, it should be noted that sequencing coverage across the entire HCV genome was consistently high, with the exception of the core protein and a portion at the N terminus of E2 (see Fig. S2 in the supplemental material). When assembled consensus NS3 sequences were compared to Sanger sequences obtained by Janssen, 98.8%, 99.6%, and 100% overall concordance in nucleotides, amino acids, and Q80K calls, respectively, were observed (see Fig. S1C in the supplemental material). The median pairwise nucleotide concordance was 99.3% (IQR, 98.1 to 99.8%) between the Janssen Sanger and BCCfE MiSeq consensus sequences, with 91.7% of the observed nucleotide differences being the result of differences in mixture calls between methods (Fig. 1). When lower-frequency variants were examined, the MiSeq method did not detect any additional samples with minority variants harboring the Q80K polymorphism with frequencies between 2 and 20%; however, a Q80R resistance variant was observed in a single sample at a prevalence of 3.3%, although the clinical significance of such a low-frequency variant is not known.
Assay precision. (i) Repeatability of PCR and sequencing.
The ability to produce repeatable results across multiple tests was examined by performing replicate testing of three representative samples per method in a single run. Twelve replicate reverse transcription, PCR amplification, and sequencing reactions were performed for each sample, starting from a single pool of extracted RNA. Intra-assay variability was extremely small, with >99.8% mean concordance observed between replicates compared to a per-sample consensus (Table 1). Only one replicate of a single sample failed to produce a sequence.
TABLE 1.
Assay repeatability was assessed using a panel of three samples per method, which were tested 12 times in a single batch
| Assaya | Sample | Success rate (no./total no. [%]) | Precision (mean ± SD) (%)b | Q80 amino acid | Q80 concordance (%)b |
|---|---|---|---|---|---|
| Primary | 51311A | 12/12 (100) | 99.9 ± 0.1 | Q | 100 |
| 51216A | 11/12 (92) | 99.9 ± 0.1 | Q | 100 | |
| 51226A | 12/12 (100) | 99.9 ± 0.1 | K | 100 | |
| Secondary | 51411A | 12/12 (100) | 100 ± 0 | Q | 100 |
| 51417A | 12/12 (100) | 99.8 ± 0.2 | Q | 100 | |
| 51418A | 12/12 (100) | 100 ± 0 | K/R | 100 |
The primary method is a 1,200-bp fragment spanning codons 1 to 400 of HCV NS3. The 564-bp secondary method is intended for use in the event of primary assay failure.
High concordance (precision) in nucleotide base calls was observed when individual sequences were compared to a per-sample consensus.
(ii) Reproducibility of PCR and sequencing.
Assay reproducibility was assessed using a panel of 11 clinically derived plasma samples for the primary method (four samples with the Q80K polymorphism and seven with wild-type Q80) and 10 plasma samples for the secondary assay (four samples with the Q80K polymorphism and six wild type). All samples were tested on five different days by two laboratory technicians. An extremely low level of interassay variability was observed across all sequenced bases in NS3; >99.7% concordance was observed between replicates in all samples tested (Table 2). No difference in Q80K interpretations were observed between replicates. All five replicates were successfully sequenced in 20 (95.2%) tested samples. The remaining sample was successfully sequenced in only 2 of 5 attempts using the 1,200-bp primary method; however, identical nucleotide sequences were obtained in each replicate.
TABLE 2.
Assay reproducibility was assessed using a panel of plasma samples, which were tested on five separate days by two laboratory technicians
| Assay | Sample | Success rate (no./total no. [%]) | Precision (mean ± SD) (%)a | Q80 amino acid | Q80 concordance (%)a |
|---|---|---|---|---|---|
| Primary | 51204A | 5/5 (100) | 100 ± 0 | K | 100 |
| 51206A | 5/5 (100) | 99.9 ± 0.1 | Q | 100 | |
| 51208A | 5/5 (100) | 99.9 ± 0.1 | Q | 100 | |
| 51210A | 5/5 (100) | 99.9 ± 0.1 | Q | 100 | |
| 51212A | 5/5 (100) | 99.7 ± 0.1 | Q | 100 | |
| 51218A | 5/5 (100) | 99.7 ± 0.2 | K/M | 100 | |
| 51220A | 5/5 (100) | 99.9 ± 0.2 | K | 100 | |
| 51222A | 5/5 (100) | 100 ± 0 | K | 100 | |
| 51224A | 5/5 (100) | 99.9 ± 0.1 | Q | 100 | |
| 51228A | 2/5 (40) | 100 ± 0 | Q | 100 | |
| 51230A | 5/5 (100) | 100 ± 0 | Q | 100 | |
| Secondary | 51411A | 5/5 (100) | 99.9 ± 0.1 | Q | 100 |
| 51412A | 5/5 (100) | 100 ± 0 | Q | 100 | |
| 51413A | 5/5 (100) | 99.7 ± 0.3 | Q | 100 | |
| 51414A | 5/5 (100) | 100 ± 0 | K | 100 | |
| 51415A | 5/5 (100) | 100 ± 0 | K | 100 | |
| 51416A | 5/5 (100) | 99.9 ± 0.1 | K | 100 | |
| 51446A | 5/5 (100) | 100 ± 0 | Q | 100 | |
| 51447A | 5/5 (100) | 99.8 ± 0.2 | Q | 100 | |
| 51448A | 5/5 (100) | 100 ± 0 | Q | 100 | |
| 51450A | 5/5 (100) | 99.9 ± 0.1 | K | 100 |
High concordance (precision) was observed when individual sequences were compared to a per-sample consensus.
Reproducibility of the MiSeq sequencing assay was assessed in a similar manner. RT-PCR and Nextera XT library preparation were attempted on 14 samples on five consecutive days. Successfully amplified libraries were sequenced on two separate MiSeq runs. Two samples failed the full-genome MiSeq assay and were excluded from the analysis of reproducibility. One sample failed to amplify in all five replicates. A PCR product was obtained for the second sample in 4 of 5 replicates; however, after sequencing and assembly, <1,000-fold coverage was obtained in the region surrounding NS3 codon 80 in all replicates. In the remaining 12 samples, no substantial differences were observed in the frequency of the Q80K polymorphism in all recovered reads across the replicates (Fig. 3). Although coverage of <1,000 reads at NS3 codon 80 was obtained in one replicate of a single sample, no effect on the detection of the Q80K polymorphism was observed.
FIG 3.
Repeatability of Q80K measurements across five independent replicates of a whole-genome HCV MiSeq sequencing assay. The MiSeq amplification protocol was attempted on 14 samples on five consecutive days. HCV NS3 was successfully amplified in all five replicates in 12 samples (85.7%). The proportion of reads in which wild-type Q80, Q80K, Q80M, or other Q80 variants was observed (y axis, log10 scale) is displayed for each successfully sequenced replicate (x axis). The dotted black line indicates the level of sequencing coverage at NS3 codon 80 in each replicate (secondary y axis, log10 scale).
Assay sensitivity and determination of the assay limit of detection.
The lower limit of detection was determined using serial dilutions (1:10) of five plasma samples (pVL range, 5.6 to 6.0 log10 IU/ml). Three samples were tested for each Sanger sequencing method, with six replicates performed at each pVL dilution. The lower limit of detection was defined in two ways: (i) PCR amplification success rate among replicates, and (ii) nucleotide sequence concordance between sequences obtained in the full-strength sample and the most dilute sample for which sequencing was successful. For simplicity, one replicate sequence from the highest and lowest concentration for each sample was selected at random for this comparison.
Using the primary Sanger method, there was 100% and 94% success in amplification and sequencing in samples with >5.0 and 4 to 5 log10 IU/ml, respectively (Table 3). The amplification and sequencing success rate was <45% in samples with a pVL of 3 to 4 log10 IU/ml; however, four of six samples with a pVL of 7,280 IU/ml were successfully amplified, suggesting a limit of detection on the order of 3.9 log10 IU/ml. Nucleotide concordance rates of 98.3 to 99.8% were observed when sequences from the lowest and highest concentrations were compared. No differences were observed at NS3 Q80 in any samples.
TABLE 3.
Estimating the lower limit of detection of HCV NS3 Q80K screening assays through serial 1:10 dilution of clinically derived plasma samples
| Assaya | Sample 1 |
Sample 2 |
Sample 3 |
Summary |
||||
|---|---|---|---|---|---|---|---|---|
| pVL (IU/ml) | No. with success/No. of reps (%) | pVL (IU/ml) | No. with success/No. of reps (%) | pVL (IU/ml) | No. with success/No. of reps (%) | pVL range (log10 IU/ml) | No. with success/No. of reps (%) | |
| Primary | ||||||||
| 1,080,000 | 6/6 (100) | >6.0 | 6/6 (100) | |||||
| 108,000 | 6/6 (100) | 728,000 | 6/6 (100) | 395,000 | 6/6 (100) | 5–6 | 18/18 (100) | |
| 10,800 | 5/6 (83) | 72,800 | 6/6 (100) | 39,500 | 6/6 (100) | 4–5 | 17/18 (94) | |
| 1,080 | 2/6 (33) | 7,280 | 4/6 (67) | 3,950 | 2/6 (33) | 3–4 | 8/18 (44) | |
| 728 | 1/6 (17) | 395 | 1/6 (17) | 2–3 | 2/12 (17) | |||
| Secondary | ||||||||
| 1,040,000 | 6/6 (100) | >6.0 | 6/6 (100) | |||||
| 104,000 | 6/6 (100) | 695,000 | 6/6 (100) | 395,000 | 6/6 (100) | 5–6 | 18/18 (100) | |
| 10,400 | 6/6 (100) | 69,500 | 6/6 (100) | 39,500 | 6/6 (100) | 4–5 | 18/18 (100) | |
| 1,040 | 6/6 (100) | 6,950 | 6/6 (100) | 3,950 | 6/6 (100) | 3–4 | 18/18 (100) | |
| 104 | 4/6 (67) | 695 | 4/6 (67) | 395 | 4/6 (67) | 2–3 | 12/18 (67) | |
PCR amplification success rates suggest that the lower limit of detection (LLOD) is >3.9 log10 IU/ml for the primary assay and >2.0 log10 IU/ml for the secondary assay.
For the secondary Sanger method, 100% success in amplification and sequencing was observed in samples with >3.0 log10 IU/ml (Table 3). In samples with a pVL of 2 to 3 log10 IU/ml, the amplification and sequencing success rate was an acceptable 67%, suggesting a limit of detection of approximately 2.0 log10 IU/ml or possibly lower. Nucleotide concordance rates of 98.6 to 99.1% were observed when sequences from the lowest and highest concentrations were compared. No differences were observed at NS3 Q80 between concentrations.
Assay specificity. (i) No PCR amplification of HCV-negative samples.
Five HCV-negative samples were tested to demonstrate that no off-target amplicons would be generated. Samples were tested in triplicate, and the results were visualized on a 1% agarose gel. As expected, no bands were observed for the negative sample sets for either the Sanger or MiSeq assay (data not shown).
(ii) No interference by potential coinfecting viruses.
HCV-positive plasma was spiked with a clinically derived HIV-1 plasma sample, an HIV-1 molecular clone, or a clinically derived HBV-positive plasma sample and subsequently processed in triplicate by the two Sanger sequencing methods. As expected, nucleotide sequences and NS3 Q80 calls were nearly identical (>99.9% concordance) between HIV/HBV-spiked and unspiked samples, suggesting no interference by potential coinfecting viruses. Similarly, amplification and sequencing of HCV NS3 were not affected by spiked-in HIV or HBV in the MiSeq assay (data not shown).
(iii) Lack of cross-reactivity with other HCV genotypes.
The ability to amplify products from non-GT1 HCV was evaluated using a commercial panel of nine plasma samples containing HCV of various genotypes: 1a, 1b, 2a/c, 2b, 3a, 4, 4a/c/d, 5a, and 6a/b. Panel samples were amplified in triplicate by both Sanger sequencing methods, and the resulting PCR amplicons were visualized on a 1% agarose gel. All replicates of HCV genotype 1a and 1b samples were successfully amplified by both methods; however, non-genotype 1 HCV samples could not be amplified by either method (data not shown).
DISCUSSION
We have developed and characterized the performance of two independent HCV genotype 1 sequencing assays for the detection of the NS3 Q80K polymorphism, which can partially compromise the efficacy of simeprevir in combination with pegylated interferon and ribavirin. Previous studies have examined the prevalence of the Q80K polymorphism in treatment-naive and treatment-experienced populations and have demonstrated a substantial disparity in the prevalence of the Q80K polymorphism in GT1-infected populations in North America versus Europe (11, 14, 21–24). We have demonstrated that both Sanger sequencing of a 1,200-bp and/or 564-bp fragment of NS3 and next-generation sequencing (MiSeq) of a near-full-genome product can be used to accurately screen for the NS3 Q80K polymorphism. The repeatability and reproducibility of all assays were extremely high, with >99.7% nucleotide concordance observed in all replicate tests. While a subset of samples was effectively clonal across the region sequenced, most samples exhibited a substantial amount of variability, as measured by the number of nucleotide mixtures called. The high level of inter- and intra-assay concordance between replicates was therefore not due to a lack of variation within the samples tested.
Our sensitivity experiments determined that the lower limit of detection is sufficiently low to allow testing in chronically HCV-infected treatment-naive patients. Samples with a pVL of >3.9 log10 IU/ml were amplified in at least two-thirds of replicates using the primary Sanger method and were sequenced with repeatable results. The secondary method appears to be capable of amplifying samples with a pVL of >2.0 log10 IU/ml, enabling it to be useful as a backup or rescue assay. The lower pVL limit of the MiSeq assay was considerably higher (∼5 log10 IU/ml), consistent with the requirement to amplify a >9-kb fragment (data not shown). Nevertheless, the lower pVL limit of either assay is below the expected viral load range of most patients being considered for simeprevir therapy (12, 13). Note that a nested-PCR strategy was chosen in order to maximize the future utility of this assay as a resistance test in DAA-treated patients in all stages of treatment, including those with very low viral copy numbers. Finally, none of the assays appear to be affected by potential coinfecting viruses (HIV or HBV).
The Sanger sequencing methods are presently in clinical use at the BC Centre for Excellence in HIV/AIDS and have been made available to interested laboratories worldwide. The RECall analysis software is freely available as a Web application (http://pssm.cfenet.ubc.ca/).
Other HCV NS3 Q80K screening assays are available as laboratory services via commercial vendors in the United States (e.g., LabCorp and Quest Diagnostics); however, limited details about assay methods and performance metrics are currently available. Like the methods presented here, these commercially available tests are validated for HCV genotype 1 only. To our knowledge, this study represents the first detailed description of the clinical validation of an HCV NS3 sequencing assay to be used for Q80K screening. It should also be noted that the methods presented here could potentially be used to screen for all major NS3 protease inhibitor resistance mutations known to date, although additional validation studies would be required.
While these methods have produced consistent and accurate results, they are not without limitations. As simeprevir is licensed only for the treatment of HCV genotype 1 infection in Canada, and the Q80K polymorphism is typically only observed in HCV GT1a, the procedures outlined in this study have been optimized for HCV genotype 1. Increased amplification and sequencing failure rates may be observed for other HCV genotypes (GT2 to 7) as a consequence of assay design. In fact, both Sanger sequencing methods failed to amplify HCV NS3 when tested against a panel of non-genotype 1 samples. It should be noted, however, that the samples in the genotype panel were diluted by the manufacturer to viral loads in the 3- to 4-log10 IU/ml range. While this is above the limit of detection of the secondary 564-bp method when used for GT1, the performance characteristics may be such that a higher pVL is required to successfully amplify non-GT1 samples. For example, the BCCfE laboratory has subsequently used these protocols to successfully amplify and sequence NS3 from high-pVL HCV GT3 samples previously misclassified as GT1 by line probe assay (LiPA) (data not shown). If necessary, these procedures could potentially be validated for use in other HCV genotypes; however, this is beyond the scope of the study presented here.
Finally, the RT-PCR process is dependent upon an adequate recovery of viral RNA during extraction and upon proper binding of the primers during amplification. Minority HCV populations carrying the Q80K polymorphism may be missed by either population sequencing method, as traditional Sanger sequencing can only detect minority variants (nucleotide mixtures) that exist in a minimum of ∼20% of the population. The MiSeq assay could potentially be used in cases in which the detection of minority resistance variants is important, although the performance of the assay in that context has not fully been evaluated. However, given the absence of minority variants carrying the Q80K polymorphism detected in this and previous studies (23, 24) and that the population prevalence of Q80K is likely due to a founder effect rather than active selection (15), deep sequencing specifically for the NS3 Q80K polymorphism may be unnecessary.
In summary, we have developed two independent assays on two sequencing platforms to screen for the HCV NS3 Q80K polymorphism. We have demonstrated excellent accuracy, repeatability, reproducibility, and sensitivity of these tests for detecting the Q80K polymorphism in samples with a pVL at or below levels expected in patients initiating simeprevir-containing regimens; in all samples in which the Q80K polymorphism was detected by an external assay, this polymorphism was detected by all three methods described here, with no false-positive results. Since these methods cover the amplification of a sufficiently large fragment of HCV NS3, the potential exists for either of these assays to be used in drug resistance testing for all currently approved NS3 protease inhibitors.
Supplementary Material
ACKNOWLEDGMENTS
This work was supported by a User Partnership Program award from Genome British Columbia. A.F.Y.P. is supported by a Career Investigator award from the Michael Smith Foundation for Health Research (MSFHR) and a New Investigator award from the Canadian Institutes of Health Research (CIHR). P.R.H. is supported by a CIHR/GlaxoSmithKline Research Chair in clinical virology.
The BC Centre for Excellence in HIV/AIDS received funding from Janssen Pharmaceuticals (the manufacturer of simeprevir). H.H. is an employee of Janssen, Inc.
Footnotes
Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00650-15.
REFERENCES
- 1.Jacobson IM, McHutchison JG, Dusheiko G, Di Bisceglie AM, Reddy KR, Bzowej NH, Marcellin P, Muir AJ, Ferenci P, Flisiak R, George J, Rizzetto M, Shouval D, Sola R, Terg RA, Yoshida EM, Adda N, Bengtsson L, Sankoh AJ, Kieffer TL, George S, Kauffman RS, Zeuzem S, ADVANCE Study Team. 2011. Telaprevir for previously untreated chronic hepatitis C virus infection. N Engl J Med 364:2405–2416. doi: 10.1056/NEJMoa1012912. [DOI] [PubMed] [Google Scholar]
- 2.Poordad F, McCone J Jr, Bacon BR, Bruno S, Manns MP, Sulkowski MS, Jacobson IM, Reddy KR, Goodman ZD, Boparai N, DiNubile MJ, Sniukiene V, Brass CA, Albrecht JK, Bronowicki J-P, SPRINT-2 Investigators. 2011. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med 364:1195–1206. doi: 10.1056/NEJMoa1010494. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Bacon BR, Gordon SC, Lawitz E, Marcellin P, Vierling JM, Zeuzem S, Poordad F, Goodman ZD, Sings HL, Boparai N, Burroughs M, Brass CA, Albrecht JK, Esteban R, HCV RESPOND-2 Investigators . 2011. Boceprevir for previously treated chronic HCV genotype 1 infection. N Engl J Med 364:1207–1217. doi: 10.1056/NEJMoa1009482. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Martell M, Esteban JI, Quer J, Genesca J, Weiner A, Esteban R, Guardia J, Gomez J. 1992. Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. J Virol 66:3225–3229. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Soriano V, Perelson AS, Zoulim F. 2008. Why are there different dynamics in the selection of drug resistance in HIV and hepatitis B and C viruses? J Antimicrob Chemother 62:1–4. doi: 10.1093/jac/dkn175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Kieffer TL, De Meyer S, Bartels DJ, Sullivan JC, Zhang EZ, Tigges A, Dierynck I, Spanks J, Dorrian J, Jiang M, Adiwijaya B, Ghys A, Beumont M, Kauffman RS, Adda N, Jacobson IM, Sherman KE, Zeuzem S, Kwong AD, Picchio G. 2012. Hepatitis C viral evolution in genotype 1 treatment-naive and treatment-experienced patients receiving telaprevir-based therapy in clinical trials. PLoS One 7:e34372. doi: 10.1371/journal.pone.0034372. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Barnard RJO, Howe JA, Ogert RA, Zeuzem S, Poordad F, Gordon SC, Ralston R, Tong X, Sniukiene V, Strizki J, Ryan D, Long J, Qiu P, Brass CA, Albrecht J, Burroughs M, Vuocolo S, Hazuda DJ. 2013. Analysis of boceprevir resistance associated amino acid variants (RAVs) in two phase 3 boceprevir clinical studies. Virology 444:329–336. doi: 10.1016/j.virol.2013.06.029. [DOI] [PubMed] [Google Scholar]
- 8.Kuntzen T, Timm J, Berical A, Lennon N, Berlin AM, Young SK, Lee B, Heckerman D, Carlson J, Reyor LL, Kleyman M, McMahon CM, Birch C, Schulze Zur Wiesch J, Ledlie T, Koehrsen M, Kodira C, Roberts AD, Lauer GM, Rosen HR, Bihl F, Cerny A, Spengler U, Liu Z, Kim AY, Xing Y, Schneidewind A, Madey MA, Fleckenstein JF, Park VM, Galagan JE, Nusbaum C, Walker BD, Lake-Bakaar GV, Daar ES, Jacobson IM, Gomperts ED, Edwards A, Donfield SM, Chung RT, Talal AH, Marion T, Birren BW, Henn MR, Allen TM. 2008. Naturally occurring dominant resistance mutations to hepatitis C virus protease and polymerase inhibitors in treatment-naïve patients. Hepatology 48:1769–1778. doi: 10.1002/hep.22549. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Bartels DJ, Sullivan JC, Zhang EZ, Tigges AM, Dorrian JL, De Meyer S, Takemoto D, Dondero E, Kwong AD, Picchio G, Kieffer TL. 2013. Hepatitis C virus variants with decreased sensitivity to direct-acting antivirals (DAAs) were rarely observed in DAA-naive patients prior to treatment. J Virol 87:1544–1553. doi: 10.1128/JVI.02294-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Bartels DJ, Zhou Y, Zhang EZ, Marcial M, Byrn RA, Pfeiffer T, Tigges AM, Adiwijaya BS, Lin C, Kwong AD, Kieffer TL. 2008. Natural prevalence of hepatitis C virus variants with decreased sensitivity to NS3.4A protease inhibitors in treatment-naive subjects. J Infect Dis 198:800–807. doi: 10.1086/591141. [DOI] [PubMed] [Google Scholar]
- 11.Palanisamy N, Danielsson A, Kokkula C, Yin H, Bondeson K, Wesslén L, Duberg A-S, Lennerstrand J. 2013. Implications of baseline polymorphisms for potential resistance to NS3 protease inhibitors in hepatitis C virus genotypes 1a, 2b and 3a. Antiviral Res 99:12–17. doi: 10.1016/j.antiviral.2013.04.018. [DOI] [PubMed] [Google Scholar]
- 12.Manns M, Marcellin P, Poordad F, de Araujo ESA, Buti M, Horsmans Y, Janczewska E, Villamil F, Scott J, Peeters M, Lenz O, Ouwerkerk-Mahadevan S, De La Rosa G, Kalmeijer R, Sinha R, Beumont-Mauviel M. 2014. Simeprevir with pegylated interferon alfa 2a or 2b plus ribavirin in treatment-naive patients with chronic hepatitis C virus genotype 1 infection (QUEST-2): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet 384:414–426. doi: 10.1016/S0140-6736(14)60538-9. [DOI] [PubMed] [Google Scholar]
- 13.Jacobson IM, Dore GJ, Foster GR, Fried MW, Radu M, Rafalsky VV, Moroz L, Craxì A, Peeters M, Lenz O, Ouwerkerk-Mahadevan S, De La Rosa G, Kalmeijer R, Scott J, Sinha R, Beumont-Mauviel M. 2014. Simeprevir with pegylated interferon alfa 2a plus ribavirin in treatment-naive patients with chronic hepatitis C virus genotype 1 infection (QUEST-1): a phase 3, randomised, double-blind, placebo-controlled trial. Lancet 384:403–413. doi: 10.1016/S0140-6736(14)60494-3. [DOI] [PubMed] [Google Scholar]
- 14.Lenz O, Verbinnen T, Fevery B, Tambuyzer L, Vijgen L, Peeters M, Buelens A, Ceulemans H, Beumont M, Picchio G, De Meyer S. 2015. Virology analyses of HCV isolates from genotype 1-infected patients treated with simeprevir plus peginterferon/ribavirin in phase IIb/III studies. J Hepatol 62:1008–1014. doi: 10.1016/j.jhep.2014.11.032. [DOI] [PubMed] [Google Scholar]
- 15.McCloskey RM, Liang RH, Joy JB, Krajden M, Montaner JSG, Harrigan PR, Poon AFY. 2015. Global origin and transmission of hepatitis C virus nonstructural protein 3 Q80K polymorphism. J Infect Dis 211:1288–1295. [DOI] [PubMed] [Google Scholar]
- 16.U.S. Food and Drug Administration. 2013. FDA Antiviral Drugs Advisory Committee meeting. Background package for NDA 205123: simeprevir (TMC435). U.S. Food and Drug Administration, Silver Spring, MD: http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/AntiviralDrugsAdvisoryCommittee/UCM371623.pdf. [Google Scholar]
- 17.Woods CK, Brumme CJ, Liu TF, Chui CKS, Chu AL, Wynhoven B, Hall TA, Trevino C, Shafer RW, Harrigan PR. 2012. Automating HIV drug resistance genotyping with RECall, a freely accessible sequence analysis tool. J Clin Microbiol 50:1936–1942. doi: 10.1128/JCM.06689-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Zhang EZ, Bartels DJ, Frantz JD, Seepersaud S, Lippke JA, Shames B, Zhou Y, Lin C, Kwong A, Kieffer TL. 2013. Development of a sensitive RT-PCR method for amplifying and sequencing near full-length HCV genotype 1 RNA from patient samples. Virol J 10:53. doi: 10.1186/1743-422X-10-53. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. doi: 10.1038/nmeth.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079. doi: 10.1093/bioinformatics/btp352. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Morel V, Duverlie G, Brochot E. 2014. Patients eligible for treatment with simeprevir in a French center. J Clin Virol 61:149–151. doi: 10.1016/j.jcv.2014.06.023. [DOI] [PubMed] [Google Scholar]
- 22.Paolucci S, Fiorina L, Piralla A, Gulminetti R, Novati S, Barbarini G, Sacchi P, Gatti M, Dossena L, Baldanti F. 2012. Naturally occurring mutations to HCV protease inhibitors in treatment-naive patients. Virol J 9:245. doi: 10.1186/1743-422X-9-245. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Jabara CB, Hu F, Mollan KR, Williford SE, Menezes P, Yang Y, Eron JJ, Fried MW, Hudgens MG, Jones CD, Swanstrom R, Lemon SM. 2014. Hepatitis C virus (HCV) NS3 sequence diversity and antiviral resistance-associated variant frequency in HCV/HIV coinfection. Antimicrob Agents Chemother 58:6079–6092. doi: 10.1128/AAC.03466-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Kirst ME, Li EC, Wang CX, Dong H-J, Liu C, Fried MW, Nelson DR, Wang GP. 2013. Deep sequencing analysis of HCV NS3 resistance-associated variants and mutation linkage in liver transplant recipients. PLoS One 8:e69698. doi: 10.1371/journal.pone.0069698. [DOI] [PMC free article] [PubMed] [Google Scholar]
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