Evolution of within-host variants of the hepatitis C virus.

INTRODUCTION: Comprehensive investigation of the within-host evolution of hepatitis C virus (HCV) variants has been difficult without high coverage deep sequencing data and bioinformatics tools to characterise these variants. With the advent of high throughput, long-read sequencing platforms such as Oxford Nanopore Technology (ONT), capturing within-host evolution of HCV using full genome sequences has become feasible. This study aimed to provide the proof of concept that within-host evolutionary analysis of HCV using near-full-length genomes, is achievable. METHODS: Five treatment naïve subjects with chronic HCV infection were sampled longitudinally from 6 months to 5 years post-infection, with 3-5 sampling timepoints per subject. Near full-length sequences generated using the ONT platform encompassing within-host HCV variants were analysed using an in-house bioinformatic tool. A 200-sequence proxy alignment of the viral variants was made for each subject and timepoint, proportionately representing the observed within-host variants. This alignment was then used in a Bayesian evolutionary analysis using BEAST software suite (v1.8). RESULTS: The estimated within-host substitution rates ranged between 0.89 and 6.19 × 10-5 substitutions/site/day. At most timepoints, observed viral lineages were closely related to those from the immediately preceding timepoint, and genetic diversity bottlenecks were observed at intervals in both the acute and chronic phases of infection. The highest within-host mutation rates were observed in the Envelope-P7 and NS5 regions while the Core region was the most conserved. CONCLUSION: This study demonstrates the feasibility of studying within-host evolution of near-full-length HCV genomes, using long-read sequencing platforms. When considered in conjunction with meta-data such as the host immune response, these methods may offer high resolution insights into immune escape (in vivo or in vitro) to inform vaccine design and to predict spontaneous clearance.

Adaptation of Oxford Nanopore technology for hepatitis C whole genome sequencing and identification of within-host viral variants.

BACKGROUND: Hepatitis C (HCV) and many other RNA viruses exist as rapidly mutating quasi-species populations in a single infected host. High throughput characterization of full genome, within-host variants is still not possible despite advances in next generation sequencing. This limitation constrains viral genomic studies that depend on accurate identification of hemi-genome or whole genome, within-host variants, especially those occurring at low frequencies. With the advent of third generation long read sequencing technologies, including Oxford Nanopore Technology (ONT) and PacBio platforms, this problem is potentially surmountable. ONT is particularly attractive in this regard due to the portable nature of the MinION sequencer, which makes real-time sequencing in remote and resource-limited locations possible. However, this technology (termed here 'nanopore sequencing') has a comparatively high technical error rate. The present study aimed to assess the utility, accuracy and cost-effectiveness of nanopore sequencing for HCV genomes. We also introduce a new bioinformatics tool (Nano-Q) to differentiate within-host variants from nanopore sequencing. RESULTS: The Nanopore platform, when the coverage exceeded 300 reads, generated comparable consensus sequences to Illumina sequencing. Using HCV Envelope plasmids (~ 1800 nt) mixed in known proportions, the capacity of nanopore sequencing to reliably identify variants with an abundance as low as 0.1% was demonstrated, provided the autologous reference sequence was available to identify the matching reads. Successful pooling and nanopore sequencing of 52 samples from patients with HCV infection demonstrated its cost effectiveness (AUD$ 43 per sample with nanopore sequencing versus $100 with paired-end short read technology). The Nano-Q tool successfully separated between-host sequences, including those from the same subtype, by bulk sorting and phylogenetic clustering without an autologous reference sequence (using only a subtype-specific generic reference). The pipeline also identified within-host viral variants and their abundance when the parameters were appropriately adjusted. CONCLUSION: Cost effective HCV whole genome sequencing and within-host variant identification without haplotype reconstruction are potential advantages of nanopore sequencing.

Single molecule, near full-length genome sequencing of dengue virus.

Current methods for dengue virus (DENV) genome amplification, amplify parts of the genome in at least 5 overlapping segments and then combine the output to characterize a full genome. This process is laborious, costly and requires at least 10 primers per serotype, thus increasing the likelihood of PCR bias. We introduce an assay to amplify near full-length dengue virus genomes as intact molecules, sequence these amplicons with third generation "nanopore" technology without fragmenting and use the sequence data to differentiate within-host viral variants with a bioinformatics tool (Nano-Q). The new assay successfully generated near full-length amplicons from DENV serotypes 1, 2 and 3 samples which were sequenced with nanopore technology. Consensus DENV sequences generated by nanopore sequencing had over 99.5% pairwise sequence similarity to Illumina generated counterparts provided the coverage was > 100 with both platforms. Maximum likelihood phylogenetic trees generated from nanopore consensus sequences were able to reproduce the exact trees made from Illumina sequencing with a conservative 99% bootstrapping threshold (after 1000 replicates and 10% burn-in). Pairwise genetic distances of within host variants identified from the Nano-Q tool were less than that of between host variants, thus enabling the phylogenetic segregation of variants from the same host.