Abstract
BackgroundViral relapse is a major concern in hepatitis C virus (HCV) antiviral therapy. Currently, there are no satisfactory methods to predict viral relapse, especially in the era of direct acting antivirals in which the virus often quickly becomes undetectable using PCR-based approaches that focus on a small viral region. Next-generation sequencing (NGS) provides an alternative option for viral detection in a genome-wide manner. However, owing to the overwhelming dominance of human genetic content in clinical specimens, direct detection of HCV by NGS has a low sensitivity and hence viral enrichment is required.MethodsBased on template-dependent multiple displacement amplification (tdMDA), an improved method for whole genome amplification (Wang et al., 2017. Biotechniques 63, 21–27), we evaluated two strategies to enhance the sensitivity of NGS-based HCV detection: duplex-specific nuclease (DSN)-mediated depletion of human sequences and HCV probe-based capture sequencing.ResultsIn DSN-mediated depletion, human sequences were significantly reduced in the two HCV serum samples tested, 65.3% → 55.6% → 33.7% (#4727) and 68.6% → 56% → 21% (#4970), respectively for no normalization, self- and driver-applied normalization. However, this approach was associated with a loss of HCV sequences perhaps due to its micro-homology with the human genome. In capture sequencing, HCV-mapped sequencing reads occupied 96.8% (#4727) and 22.14% (#4970) in NGS data, equivalent to 1936x and 7380x enrichment, respectively. Capture sequencing was then applied to ten serum samples collected at the end of HCV antiviral therapy. Interestingly, the number of HCV-mapped reads was significantly higher in relapsed patients (n = 5) than those from patients with sustained virological response (SVR) (n = 5), 102.4 ± 72.3 vs. 2.6 ± 0.55, p = 0.014.ConclusionsOur data provides concept evidence for a highly sensitive HCV detection by capture sequencing. The abundance difference of HCV sequencing reads at the end of HCV antiviral therapy could be applied to predict treatment outcomes.
Highlights
Viral relapse is a major concern in hepatitis C virus (HCV) antiviral therapy
A drawback of using Next-generation sequencing (NGS) for viral detection is its low sensitivity owing to the overwhelming dominance of human and commensal microbial genomes in clinical specimens [8]
Enrichment comparison: duplex-specific nuclease (DSN)-mediated depletion vs. capture sequencing Following reverse transcription (RT)-template-dependent multiple displacement amplification (tdMDA), samples #4727 and #4970 were subjected to Illumina sequencing under three options, regular library preparation, DSN-mediate depletion of human genomic sequences prior to library construction, and capture hybridization
Summary
Viral relapse is a major concern in hepatitis C virus (HCV) antiviral therapy. Currently, there are no satisfactory methods to predict viral relapse, especially in the era of direct acting antivirals in which the virus often quickly becomes undetectable using PCR-based approaches that focus on a small viral region. Patients at the time of HCV relapse should maintain a low-level viral replication that is beyond the detection limit of current PCR-based methods [7]. In this setting, an alternative option is next-generation sequencing (NGS) that may detect the virus in a genome-wide manner. Our lab previously developed template-dependent multiple displacement amplification (tdMDA) that eliminates primer-mediated artifacts [9] Based on this improved whole genome amplification method, the current study attempted to increase the sensitivity of NGS through two enrichment strategies, depletion of human sequences or target capture by probe-based hybridization. The optimized method was applied to clinical serum samples to explore the feasibility in detecting extremely low viral titers, thereby providing an on-treatment tool in predicting the outcomes of HCV antiviral therapy
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