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World J Gastroenterol. Aug 28, 2019; 25(32): 4661-4672
Published online Aug 28, 2019. doi: 10.3748/wjg.v25.i32.4661
Exploring the hepatitis C virus genome using single molecule real-time sequencing
Haruhiko Takeda, Taiki Yamashita, Yoshihide Ueda, Akihiro Sekine
Haruhiko Takeda, Taiki Yamashita, Akihiro Sekine, Department of Omics-based Medicine, Center for Preventive Medical Science, Chiba University, Chiba 260-0856, Japan
Haruhiko Takeda, Yoshihide Ueda, Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
Author contributions: Takeda H and Yamashita T contributed to literature review and drafting of the manuscript; Ueda Y and Sekine A contributed to critical revision and editing of the manuscript; all authors approved the final version of the manuscript.
Conflict-of-interest statement: No potential conflicts of interest. No financial support was received for this work.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Akihiro Sekine, PhD, Professor, Department of Omics-based Medicine, Center for Preventive Medical Science, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba 260-0856, Japan. sekine.akihiro@chiba-u.jp
Telephone: +81-226-2537
Received: May 17, 2019
Peer-review started: May 17, 2019
First decision: June 16, 2019
Revised: July 4, 2019
Accepted: July 19, 2019
Article in press: July 19, 2019
Published online: August 28, 2019
Processing time: 104 Days and 5.5 Hours
Abstract

Single molecular real-time (SMRT) sequencing, also called third-generation sequencing, is a novel sequencing technique capable of generating extremely long contiguous sequence reads. While conventional short-read sequencing cannot evaluate the linkage of nucleotide substitutions distant from one another, SMRT sequencing can directly demonstrate linkage of nucleotide changes over a span of more than 20 kbp, and thus can be applied to directly examine the haplotypes of viruses or bacteria whose genome structures are changing in real time. In addition, an error correction method (circular consensus sequencing) has been established and repeated sequencing of a single-molecule DNA template can result in extremely high accuracy. The advantages of long read sequencing enable accurate determination of the haplotypes of individual viral clones. SMRT sequencing has been applied in various studies of viral genomes including determination of the full-length contiguous genome sequence of hepatitis C virus (HCV), targeted deep sequencing of the HCV NS5A gene, and assessment of heterogeneity among viral populations. Recently, the emergence of multi-drug resistant HCV viruses has become a significant clinical issue and has been also demonstrated using SMRT sequencing. In this review, we introduce the novel third-generation PacBio RSII/Sequel systems, compare them with conventional next-generation sequencers, and summarize previous studies in which SMRT sequencing technology has been applied for HCV genome analysis. We also refer to another long-read sequencing platform, nanopore sequencing technology, and discuss the advantages, limitations and future perspectives in using these third-generation sequencers for HCV genome analysis.

Keywords: Third generation sequencing; PacBio RSII; Single molecule real-time sequencing; Hepatitis C virus; Resistance-associated substitution; Nanopore sequencer

Core tip: Single molecular real-time (SMRT) sequencing, also called third-generation sequencing, is a novel sequencing technique capable of generating extremely long contiguous sequence reads. The advantages of long read sequencing enable accurate determination of the haplotypes of infected viral clones. We introduce the novel third-generation sequencers PacBio RSII/Sequel systems, compare them with conventional next-generation sequencers, and summarize previous studies in which SMRT sequencing technology has been applied for hepatitis C virus genome analysis.