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New sequencing technology, third‐generation nanopore sequencing, for hepatitis C virus research

Progress in sequencing technology has changed our understanding of the landscape of a hepatitis C virus (HCV) infection, which shows abundant diversity and complexity in a patient with chronic hepatitis C. In this issue of Hepatology Research, Uchida et al. report a case of genotype 3b HCV analyzed by a new sequencing technology, nanopore sequencing. The authors showed the usefulness of the nanopore sequencing technique for identifyingHCV sequence at low cost and completing analysis within a short duration. Nanopore sequencing is also referred to as single molecule real-time (SMRT) sequencing, and can generate extremely long contiguous sequence reads. This technique is also called third-generation sequencing, distinguishing it from next-generation sequencing (NGS) methods, which are carried out using Illumina (San Diego, CA, USA) MiSeq, HiSeq, or Ion Torrent sequencers. Nextgeneration sequencing can detect rare nucleotide variants with variant frequencies of less than 1%, using deep sequencing methods that generate sequence reads with much higher throughput than Sanger sequencing, which can read only a few major clones in an infected individual with an enormous HCV viral load (Figure 1). Therefore, NGS has enabled us to detect rare variants with resistance-associated substitutions (RAS) to direct-acting antivirals (DAA) in the sera of patients infected with HCV. A major limitation of NGS technologies for analysis of multidrug resistant variants is its short read lengths (~400 bp). Hepatitis C virus has a single-stranded 9.6-kb RNA genome, and several RAS are known to distribute within the non-structural protein (NS)3 to NS5B region (Figure 1). Although NGS can show the frequency of each variant within a sequenced sample, it cannot evaluate the linkage of multiple nucleotide variants, which are far apart from each other in the same viral clone (haplotype). As a result, short-read NGS technologies cannot evaluate multidrug resistant viral clones, which are closely related to relapse during or after DAA therapy. To overcome this limitation of NGS, two types of thirdgeneration automated sequencers have recently been developed, the nanopore sequencer (OxfordNanopore Technologies, Oxford, UK) and the PacBio sequencer (Pacific Biosciences, Menlo Park, CA, USA). Both sequencers use the SMRT sequencing technique, which allows them to generate extremely long contiguous sequence reads. The long-read sequencing ability of third-generation sequencers makes it possible to determine the haplotype of individual HCV clones and clarify the linkage of RAS distributed from NS3 to NS5B (Fig. 1). The two SMRT techniques use different methods of sequencing, which have distinguishable characteristics. The characteristics of the PacBio sequencer include high accuracy conferred by circular consensus sequencing methods. Reading an identical template 10 times reduces the error rate to as low as 0.03%. In fact, Takeda et al. determined long contiguous sequences spanning NS3 toNS5A regions of eachHCV clone inpatients’ sera byusing a PacBio sequencerwith extremely high accuracy of sequence reads. However, due to the limited throughput and high costs of the PacBio system,many small laboratories cannot access this instrument to date. The nanopore sequencer uses completely different technologies from the PacBio sequencer. The nanopore sequencer directly detects the DNA composition of a native single-stranded DNA molecule passing through a protein pore by the change in electronic voltage. No polymerase reaction is used in this sequencing process. This novel method allows the nanopore sequencer to achieve low cost, require only USB power, be produced on a handheld Hepatology Research 2019; 49: 961–963 doi: 10.1111/hepr.13383