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

BackgroundDeep 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.AimGenotype-independent high-throughput pyrosequencing was used to obtain near full length hepatitis C virus genome sequence reconstruction directly from clinical samples.MethodsSamples from hepatitis C virus infected subjects harbouring different subtypes (1a, 1b, 2c) were analyzed (viral load range: 1.2–20.8 × 106 IU/ml). Data were generated with a modified sequence-independent single primer amplification method followed by 454 sequencing.Resultsthe 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.ConclusionThis study describes a protocol for obtaining whole genome sequences from different hepatitis C virus patients with different genotypes in a single sequencing run.