Comparison of next Generation Sequencing Technologies for the Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes

Abstract

f Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of highthroughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.

Presence of Human Hepegivirus-1 in a Cohort of People Who Inject Drugs

“…In clinical practice, NGMS has already been used for detecting unsuspected pathogens (7,9,10). In addition, NGMS is being used to characterize the composition of complex populations of already-recognized viruses like human immunodeficiency virus −1 (HIV) and hepatitis C virus (HCV) (so called, 'deep sequencing') (11,12). However, while NGMS can uncover novel sequences, the limits of detection are not clearly defined.…”

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

“…Oligonucleotide bait probes have also shown promise as an alternative to metagenomics and amplicon sequencing 39–42 . The method fish for viral nucleic acid sequences by hybridising target-specific biotinylated probes to the DNA sample then pull them down using magnetic streptavidin beads.…”

Highly Diverse Hepatitis C Strains Detected in Sub‐Saharan Africa Have Unknown Susceptibility to Direct‐Acting Antiviral Treatments

“…The newer pangenotypic combination therapies sofosbuvir, voxilaprevir, and velpatasvir and glecaprevir and pibrentasvir are less likely to fail in the presence of mutations such as Y93H in other genotypes, so these may prove to be effective; however, no studies in g7 have yet been carried out. g7 HCV has been shown to be mistyped as g2 in other studies using the TRUGENE assay; this could result in errors in therapeutic decision making, and the adoption of full genome sequencing using more sophisticated technologies such as metagenomic and target enrichment–based NGS may reveal other “rare” genotypes as a cause of treatment failure as treatment is rolled out . Unbiased metagenomic NGS sequencing overcomes the need for specific primers for full genome sequencing and may therefore help to identify new strains that would not have been amplified using PCR‐based methodology.…”

“…This was carried out using a metagenomic sequencing protocol as described . Briefly, RNA was extracted from 200 µL of plasma using the Agencourt RNAdvance Blood Kit (Beckman Coulter) and reverse transcribed using SuperScript III (Invitrogen) with random hexamers and an NEB Second Strand Synthesis Kit (New England Biolabs).…”