Hepatitis C virus whole genome sequencing: Current methods/issues and future challenges

Background: Injecting drugs substantially increases the risk of hepatitis C virus (HCV) infection and is common in vulnerable population groups, such as the homeless and prisoners. Capturing accurate data on relative genotype distribution within these groups is essential to inform strategies to reduce HCV transmission. The aim of this study was to utilise a next-generation whole-genome sequencing method recently validated by Public Health England, in order to produce near complete HCV genomes. Methods: In total, 98 HCV positive patients were recruited from homeless hostels and drug treatment services through the National Health Services (NHS) Find and Treat (F&T) Service between May 2011 and June 2013 in London, UK. Samples were sequenced by Next-generation sequencing, with 88 complete HCV genomes constructed by a de novo assembly pipeline. They were analysed phylogenetically for an estimate of their genetic distance. Results: Of the 88 complete HCV genomes, 50/88 (56.8%) were genotype 1; 32/88 (36.4%) genotype 3; 4/88 (4.5%) genotype 2; and 1/88 (1.1%) for genotypes 4 and 6 each. Subtype 1a had the highest number of samples (51.1%), followed by subtype 3a (35.2%), 1b (5.7%), and 2b (3.4%). Samples collected from drug treatment services had the highest number of genotype 1 (69%); genotypes 4 and 6 were only found from samples collected in homeless shelters. Small clusters of highly related genomic sequences were observed both across and within the vulnerable groups sampled. Conclusions: Subsequent phylogenetic analysis provides a first indication that there are related HCV sequences amongst the three vulnerable population groups, reflecting their overlapping social behaviours. This study is the first presentation of whole genome HCV sequences from such vulnerable groups in London and paves the way for similar research in the future.

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

BMC Genomics ◽

10.1186/s12864-021-07460-1 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Nasir Riaz ◽

Preston Leung ◽

Kirston Barton ◽

Martin A. Smith ◽

Shaun Carswell ◽

...

Keyword(s):

Cost Effectiveness ◽

Hepatitis C ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Reference Sequence ◽

Whole Genome ◽

Nanopore Sequencing ◽

Accurate Identification ◽

Low Frequencies ◽

Oxford Nanopore

Abstract 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.

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