Near full length hepatitis C virus genome reconstruction by next generation sequencing based on genotype-independent amplification

Abstract

Background: Deep sequencing has a deep impact on the study of rapidly mutating RNA viruses, such as hepatitis C virus, proving to be an invaluable tool for analyzing virus diversity and evolution.

Aim: Genotype-independent high-throughput pyrosequencing was used to obtain near full length hepatitis C virus genome sequence reconstruction directly from clinical samples.

Methods: Samples from hepatitis C virus infected subjects harbouring different subtypes (1a, 1b, 2c) were analyzed (viral load range: 1.2-20.8 × 10(6)IU/ml). Data were generated with a modified sequence-independent single primer amplification method followed by 454 sequencing.

Results: the extent of reconstructed hepatitis C virus genome varied from 79.95% to 99.64%. No correlation between extent of genome reconstruction and either viral load (r=0.4857, p=0.3556) or number of HCV reads (r=0.08571, p=0.9194) was observed.

Conclusion: This study describes a protocol for obtaining whole genome sequences from different hepatitis C virus patients with different genotypes in a single sequencing run.

Keywords: DAAs; Deep-sequencing; Full genome reconstruction; HCV; Modified SISPA approach.