Exploring the hepatitis C virus genome using single molecule real-time sequencing

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.

Single-molecular real-time deep sequencing reveals the dynamics of multi-drug resistant haplotypes and structural variations in the hepatitis C virus genome

“…These sequencing technologies generate reads of over 10 kbp 22,23 . While the accuracy of the raw sequence reads from third-generation sequencers is limited to 90%, a characteristic error correction methodology by the PacBio sequencer system, called circular consensus sequencing (CCS) technology, improves the accuracy of the raw reads to as high as 99.9% 24 . Due to these characteristics, the PacBio RS II/Sequel can produce high quality sequence reads of HCV genomes at the single-molecule level, including multiple RASs, rare RASs, and SVs.…”

Characterization and analysis of full-length transcriptomes from two grasshoppers, Gomphocerus licenti and Mongolotettix japonicus

“…Read length is an important advantage of PacBio sequencing. The read length generated by PacBio RSII system ranged from 5 to 60 kb, with an average length around 12 kb, and the newly-released PacBio Sequel sequencer can generate reads longer than 20 kb on average, which is about 200 times longer than those reads generated by conventional NGS instruments 11 . Due to the much longer read lengths of PacBio sequencing, the precise locations and sequences of repetitive regions and isoforms can often be resolved with a single read.…”

Whole Genome Sequencing of Hepatitis A Virus Using a PCR-Free Single-Molecule Nanopore Sequencing Approach

“…Some of the limitations to obtain complete genomes using high-throughput sequencing (HTS) are mainly due to the shorter reads produced. Short reads not only can impair the detection of different viral haplotypes (Takeda et al, 2019) and introduce ambiguities in de novo assemblies (Schatz et al, 2010) but are also unable to resolve complex regions of the genome (Somerville et al, 2019;Treangen and Salzberg, 2011). Additionally, the PCR amplification steps involved during library preparation can introduce considerable bias, for example selective amplification and chimeras (Ardui et al, 2018;Ng et al, 2018).…”

“…Nanopore sequencing provides genomelength reads in real time, generating an integral vison of the genome that includes all possible mutations within a single virus particle (McNaughton et al, 2019). The particularly long reads generated by nanopore sequencing allow the complete genome to be sequenced from single viral clones and consequently make possible the detection of undescribed or rare variants (Takeda et al, 2019). The use of nanopore sequence data will increase, without precedent our ability to characterize mutant haplotypes, which reflect the immense genetic diversity of viral related sequences (aka quasispecies) within infected cells and host organisms (Andino and Domingo, 2015).…”

“…This will be important to further extend our understanding of sequence variants underlying phenotype and disease. Recently nanopore sequencing was used for whole-genome sequencing of the Hepatitis C virus (Takeda et al, 2019;Ueda, 2019) and Hepatitis B virus (McNaughton et al, 2019). Nanopore sequencing was also used to serotype the Foot-and-mouth disease virus, which also belongs to the Picornaviridae family (Hansen et al, 2019).…”