Comparison of Next-Generation Sequencing and Clone-Based Sequencing in Analysis of Hepatitis B Virus Reverse Transcriptase Quasispecies Heterogeneity

Abstract

We previously reported that, based on clone-based sequencing (CBS), hepatitis B virus (HBV) heterogeneity within the reverse transcriptase (RT) region was a predictor of antiviral efficacy. Here, by comparing ultradeep pyrosequencing (UDPS), i.e., nextgeneration sequencing (NGS), with CBS in characterizing the genetic heterogeneity of HBV quasispecies within the RT region, we evaluated the performance of UDPS in the analysis of HBV viral populations. HBV genomic DNA was extracted from serum samples from 31 antiviral treatment-naive patients with chronic hepatitis B. The RT region quasispecies were analyzed in parallel using CBS and UDPS. Characterization of quasispecies heterogeneity was conducted using bioinformatics analysis. Quasispecies complexity values were calculated with the formula Sn ‫؍‬ ؊⌺ i (p i lnp i )/lnN. The number of qualified strains obtained by UDPS was much larger than that obtained by CBS (P < 0.001). Pearson analysis showed that there was a positive correlation of quasispecies complexity values at the nucleotide level for the two methods (P < 0.05), while the complexity value derived from UDPS data was higher than that derived from CBS data (P < 0.001). Study of the prevalences of variations within the RT region showed that CBS detected an average of 9.7 ؎ 1.1 amino acid substitutions/sample and UDPS detected an average of 16.2 ؎ 1.4 amino acid substitutions/sample. The phylogenetic analysis based on UDPS data showed more genetic entities than did that based on CBS data. Viral heterogeneity determination by the UDPS technique is more sensitive and efficient in terms of lowabundance variation detection and quasispecies simulation than that by the CBS method, although imperfect, and thus sheds light on the future clinical application of NGS in HBV quasispecies studies. Hepatitis B virus (HBV) is a noncytopathic DNA virus that infects approximately 350 million people worldwide and is a leading cause of liver cirrhosis and hepatocellular carcinoma. The evolutionary dynamics of HBV are characterized by high mutation rates (Ͼ10 Ϫ5 nucleotide substitutions/site/year) due to the error-prone reverse transcriptase (RT) (1) and rapid replication rates (Ͼ10 11 virions per day) (2, 3). Thus, a large number of viral variants are constantly generated, creating great genetic diversity on which natural selection operates. Heterogeneous populations of viruses are often referred to as viral quasispecies (4, 5). The massive heterogeneity present in HBV quasispecies has important biological consequences; it has been identified as an important factor in relation to the clinical features of the infection (transmission, persistence, and liver damage), and it may influence the outcomes of treatment and be important in the development of resistance to nucleoside/nucleotide analogue (NA) antiviral therapy (6).Cloning of PCR products and subsequent Sanger dideoxy sequencing have been widely used in analyses of the heterogeneity of viral quasispecies and determination of antiviral-associated drug resistance mutations, ...

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