Detection of low-level HCV variants in DAA treated patients: comparison amongst three different NGS data analysis protocols
Caputo et al. Virology Journal (2020) 17:103
Background: Notwithstanding the efforts of direct-acting antivirals (DAAs) for the treatment of chronically infected
hepatitis C virus (HCV) patients, concerns exist regarding the emergence of resistance-associated substitutions (RAS)
related to therapy failure. Sanger sequencing is still the reference technique used for the detection of RAS and it
detects viral variants present up to 15%, meaning that minority variants are undetectable, using this technique. To
date, many studies are focused on the analysis of the impact of HCV low variants using next-generation sequencing
(NGS) techniques, but the importance of these minority variants is still debated, and importantly, a common data
analysis method is still not defined.
Methods: Serum samples from four patients failing DAAs therapy were collected at baseline and failure, and
amplification of NS3, NS5A and NS5B genes was performed on each sample. The genes amplified were sequenced
using Sanger and NGS Illumina sequencing and the data generated were analyzed with different approaches. Three
different NGS data analysis methods, two homemade in silico pipeline and one commercially available certified
user-friendly software, were used to detect low-level variants.
Results: The NGS approach allowed to infer also very-low level virus variants. Moreover, data processing allowed to
generate high accuracy data which results in reduction in the error rates for each single sequence polymorphism.
The results improved the detection of low-level viral variants in the HCV quasispecies of the analyzed patients, and
in one patient a low-level RAS related to treatment failure was identified. Importantly, the results obtained from
only two out of the three data analysis strategies were in complete agreement in terms of both detection and
frequency of RAS.
Conclusions: These results highlight the need to find a robust NGS data analysis method to standardize NGS results
for a better comprehension of the clinical role of low-level HCV variants. Based on the extreme importance of data
analysis approaches for wet-data interpretation, a detailed description of the used pipelines and further standardization
of the in silico analysis could allow increasing diagnostic laboratory networking to unleash true potentials of NGS.