Comparison of pyrosequencing and sanger sequencing for HBV DNA genotyping and resistance mutations

Drug resistance gene mutations in Hepatitis B virus (HBV) are the main reason for failure of currently used therapeutic nucleoside analogues. Two methods-Pyrosequencing and Sanger sequencing, are most commonly used for HBV genotyping and identification of its mutations, but their advantages of the two methods are undefined. Herein, the two methods were used to identify the HBV genotypes and drug-resistance mutations in the sera specimen of 138 HBV patients treated with nucleoside analogues. It had no significant difference in the detective rate of HBV genotypes B or C between the two methods, but the Pyrosequencing had an error rate of 7.25% for HBV genotyping but Sanger sequencing showed no mistakes. Sanger sequencing also had a lower failure rate and a significantly higher detection rate for the common drug-resistance mutations of HBV compared with the Pyrosequencing, and it could detect unknown new mutations in clinical samples. We also found that the Sanger sequencing had significant higher detection rate for single and multiple drug resistance mutations than the Pyrosequencing. In summary, the results indicated that the Sanger sequencing is a more reliable method with a lower failure rate and a higher detection rate for drug-resistance mutations in HBV patients’ samples, particularly in that with long-term anti-virus treatment.