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

Abstract

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 high-throughput 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 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.…”

Rapid Metagenomic Next-Generation Sequencing during an Investigation of Hospital-Acquired Human Parainfluenza Virus 3 Infections

“…The main limitation of the approach taken here is the reduced sensitivity of mNGS relative to that of PCR (24). mNGS is often unable to recover whole genomes from FilmArray-positive specimens, as illustrated in this study.…”

Current Trends in Diagnostics of Viral Infections of Unknown Etiology

“…Unlike traditional tests [54], metagenomics removes the necessity of designing and synthesizing PCR primers and probes. This reduces time consumption, which is critical during outbreaks of viral infections, such as Zika virus [55] or Ebola virus [56], when fast unbiased pathogen identification is crucial for effective disease containment.…”

“…High content of a host's nucleic acids might artificially lower CPC, falsely attributing negative results to samples. As we have mentioned, the sequencing depth is another aspect to this issue, because shallow sequencing provides little data with poor scalability, to the point where an extra round of sequencing is required to verify the results based on low SRPS data [54]. Therefore, taking the aforementioned factors into account and designing a functional SRPS assessment tool poses an important objective for further research.…”