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HCV genotyping by next generation sequencing

Hepatitis C virus (HCV) is a globally prevalent and genetically diverse virus that infects approximately 170 million people worldwide (approximately 3% of the world population).1 Of these, 55–85% will develop chronic hepatitis that in about 30% will later evolve to cirrhosis, and hepatocarcinoma in 2%.2 Chronic HCV infection is one of leading causes of liver-related death and in many countries it is a primary reason for liver transplantation. HCV is estimated to be responsible for 350,000–500,000 deaths per year.3 Since its discovery in 1989, research in molecular biology has resulted in a greater understanding of HCV, due to the difficulties in cell cuture of the virus. HCV is an enveloped, positive-sense single-stranded RNA virus of the Flaviviridae family (genus Hepacivirus), with an RNA genome of 9.6kb. Its single open-reading frame codes for ten structural and non-structural proteins; non-coding (NC) regions flank both sides and display significant secondary structures4 (Figure 1).
HCV is classified into seven major genotypes based on phylogenetic analysis, with an average of 35% nucleotide divergence between strains belonging to different genotypes. Each genotype can be further subdivided into related subtypes (67 confirmed), with a nucleotide sequence divergence of 15–30%.5 The seven genotypes vary in worldwide distribution and each are associated with differences in disease severity.6 HCV infection is a highly dynamic process because the virus has a half-life of only a few hours and it is estimated that up to 1012 new viral particles are generated per day in a patient with chronic infection. In the replication cycle, the synthesis of the viral genome is performed by a viral NS5B RNA-dependent RNA polymerase, lacking any proof-reading mechanisms. This causes a mutation rate per nucleotide of approximately 10-3 base substitutions per site per year, thus resulting in one mutation for every copied genome.7 This inaccurate replication mechanism, coupled with the high rate of replication, results in HCV isolates having extremely high sequence diversity. Genetic recombination between different strains, infecting the same patient and present in the same cell, is another source of variability. Consequently, HCV in the individual is present not as a single species, but also in the form of ‘quasi-species’ or as a heterogeneous population of virions.8
HCV treatment
The lack of detailed information about the viral replication cycle has significantly contributed to prevent the development of direct antiviral drugs. For decades chronic HCV therapy was uniquely based on interferon (IFN), which was then combined with ribavirin (RBV). However, this therapeutic approach did not achieve satisfying outcomes due to the high genetic variability of the virus. Genotypes 2 and 3 are more responsive to treatment with IFN and RBV, whereas genotypes 1 and 4 show the highest level of resistance to these drugs. In particular, genotype 1b is linked to the most acute liver inflammation, and has the worst clinical outcome.9
Direct-acting antiviral agents (DAAs) are now available. There are currently three major classes of DAAs approved for the treatment of HCV. These include inhibitors of the HCV NS3-4A protease, NS5A protein and NS5B polymerase (nucleoside analogs and non-nucleoside inhibitors). 
There are numerous DAAs in development. These newer molecules are hoped to be more active and have broader genotypic coverage.10 They are currently used in oral, IFN-free combinations for three to six months, with or without RBV.11 DAAs are effective in curing about 90% of patients with few side effects. With more molecules in various clinical trial stages, updates in recommendations for HCV treatment are anticipated in the near future. Yet, this change of therapeutic strategy alone is insufficient. Increased efficacy of therapy will not have any significant impact on HCV-related disease burden, unless combined with improvement of treatment uptake, adherence, and therefore an increased awareness of the problem.12
Genotype identification
Sequencing the HCV genome is essential for important applications, such as genotyping a patient’s strains in order to choose the most effective therapy, searching for resistance-associated mutations or studying the natural history and evolution of viral populations within one single infectious episode or on a larger epidemiological bases. Genotype identification is long established in clinical practice, because different genotypes have different response rates and require different doses and durations of IFN/RBV treatment. Moreover, co-infection with two or more HCV strains of different genotypes or subtypes is a common finding in some high-risk population groups.13,14 For this reason, it is necessary for accurate HCV subtyping because it is a fundamental tool to optimise present and future clinical management. Depending on the research subject, sequencing can be focused on different genomic regions. 
Genotyping has been mainly performed focusing the 5’ non-coding region (5’NC), the core region and the NS5B region. This is currently mandatory before treatment due to the variable response to antivirals of the different HCV types.15 Most of the commercially available genotyping methods are based on the detection of the conserved bases within the 5’ untranslated region (5’UTR). Currently, genotyping methods are based on reverse hybridisation, targeting the 5’UTR and core regions (LiPA) and real-time polymerase chain reaction (RT-PCR) assays targeting 5’UTR and NS5B that are capable of identifying major HCV genotypes in the majority of cases. These techniques are widely used because of their technical simplicity. However, these methods have limited subtyping accuracy (with the exception of genotypes 1a and 1b identification) and they have not been designed to confidently and efficiently identify mixed infections.
The recent development of different classes of DAAs targeting the NS3, NS5A and NS5B proteins has led to the sequencing of the respective genomic regions. Therapeutic pressure may lead to selection of resistance-associated variants among quasi-species, and it is necessary to look for these mutations in the case of every treatment failure. The usefulness of detecting these polymorphisms prior to therapy, however, remains unclear.16 Recently alternative regions have been proposed for HCV genotyping; the most widely accepted reference method for HCV genotyping is based on the sequencing of NS5B, the viral gene codifying for viral RNA-dependent RNA polymerase. 
Genotyping by Ion Torrent technology 
The Sanger sequencing method for the NS5B genomic region has long been considered the ‘gold standard’ for proper HCV genotyping. A major change occurred in the 2000s with the introduction of ‘next-generation sequencing’ (NGS), which provides high sequencing throughput. The main technologies are the Roche 454, Illumina, Life Technologies Ion Torrent and SOLiD. Each one has different characteristics in terms of read length, throughput and cost. Technological progress has led to continual improvements in NGS and the current development of third generation sequencing.17 Ion Torrent Genotyping Assay workflow starts with the extraction and purification, via magnetic beads technology, of nucleic acids. After extraction, NS5B is amplified via RT-PCR. PCR products are then normalised via magnetic beads to obtain an even concentration of PCR fragments. Because of the defined concentration of beads, only a defined concentration of PCR fragments will bind, and surplus fragments are washed away by a magnetic separator. After normalisation, the PCR fragments are cut into smaller pieces (200bp), to which adapters are linked to create the library. The library is then clonally amplified by emulsion PCR. 
Ideally the PCR products are clonal beads population; polyclonal and empty beads will be filtered out. Monoclonal beads are sequenced using Ion Torrent technology. The sequencing chemistry is remarkably simple. Naturally, a proton is released when a nucleotide is incorporated by the polymerase in the DNA molecule, resulting in a detectable local change of pH. Each micro-well of the Ion Torrent semiconductor sequencing chip contains approximately one million copies of a DNA molecule (Figure 2). 
Figure 2: Ion 318TM chip
The Ion Torrent sequentially floods the chip with one nucleotide after another. If a nucleotide complements the sequence of the DNA molecule in a particular micro-well, it will be incorporated and hydrogen ions are released. As a consequence, the pH of the solution changes in that well and is detected by the ion sensor, essentially going directly from chemical information to digital information. All sequenced reads are mapped to a reference genome; unmapped reads and reads mapping to non-HCV reference are excluded from analysis. The NS5B contigs are used to determine genotype using a phylogenetic tree approach.18 The strength of NGS technology is the capability of its higher resolution in detecting sequences representing <1% of the total population. Sanger sequencing, due to the low sensitivity (20%) towards minority variants present in the viral population, is unable to highlight the presence of mixed infections;19in this respect, NGS is the method of choice, due to the massive output of clonal sequence data that allows an in depth analysis of complex viral populations present in each single sample to be performed. A study based on 207 samples sequenced by Ion Torrent NGS highlights and confirms the higher sensitivity and performance in subgenotyping HCV with respect to widely used methods. Findings show, for 30 HCV genotype-2 samples, NGS assigned 30/30 (100%) subtypes, while commercial assay (LiPA) assigned only 1/30 (3.33%). In relation to HCV genotype 4, NGS assigned 14/14 subtypes (100%), whereas the other method was not able to assign any subtype.
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15 European Association for the Study of the Liver. EASL recommendations on treatment of hepatitis C. J Hepatol 2015;63:199–236.
16 Lontok E et al. Hepatitis C virus drug resistance-associated substitutions: State of the art summary. Hepatology 2015;62:1623–32.
17 Trémeaux P et al. Hepatitis C virus whole genome sequencing: Current methods/issues and future challenges. Crit Rev Clin Lab Sci 2016;53(5):341–51.
18 Application Note by Ion Torrent Life Technologies (ThermoFisher Scientific).
19 Capobianchi MR et al. Next-generation sequencing technology in clinical virology. Clin Microbiol Infect 2013;19:15–22.