Analysis of hepatitis B virus drug-resistant mutant haplotypes by ultra-deep pyrosequencing

Abstract

Direct sequencing and reverse hybridization are currently the main methods for detecting drug-resistance mutations of hepatitis B virus (HBV). However, these methods do not enable haplotype analysis so they cannot be used to determine whether the mutations are co-located on the same viral genome. This limits the accurate identification of viral mutants that are resistant to drugs with a high genetic barrier. In our current study, ultra-deep pyrosequencing (UDPS) was used to detect HBV drug-resistance mutations in 25 entecavir-treated and five treatment-naive patients. Of the 25 entecavir-treated patients, 18 had experienced virological breakthrough and two exhibited reduced susceptibility to entecavir. The results obtained by UDPS were compared with those of direct sequencing, and the haplotypes of the drug-resistant HBV mutants were analysed. The average number of reads per patient covering the region in which drug-resistance mutations are located was 1735 (range 451-4526). UDPS detected additional drug-resistance mutations not detected by direct sequencing in 19 patients (mutation frequency range 1.1-23.8%). Entecavir-resistance mutations were found to be co-located on the same viral genome in all 20 patients displaying virological breakthrough or reduced susceptibility to entecavir. In conclusion, UDPS was not only sensitive and accurate in identifying drug-resistance mutations of HBV but also enabled haplotype analysis of the mutants. This method may offer significant advantages in explaining and predicting the responses of patients with HBV to antiviral therapy.

Unraveling the complexity of tyrosine kinase inhibitor–resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain

“…Because (1) several studies concordantly demonstrated high reliability and reproducibility of 454 technology for variants >1%, [18][19][20] and (2) serial dilution experiments (see supplemental Methods for details) confirmed the ability of our assay to detect mutations as low as 1% (whereas lower levels were not investigated), we decided to reduce the likelihood of false-positive results by filtering-out variants with ,1% abundance and to exploit high coverage only for haplotype and clonal evolution analyses.…”

High-Throughput Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry as an Alternative Approach to Monitoring Drug Resistance of Hepatitis B Virus

“…The newly available ultradeep pyrosequencing (UDPS) technique, currently the most competitive for mass spectrometry, detects HBV drug-resistant strains in plasma samples if they show HBV DNA levels Ն10 3 IU/ml, which is 10 times higher than those detected using MALDI-TOF MS. Moreover, several drug resistance mutations were detected at frequencies of Ͼ1% (40,41). Thus, high-throughput MALDI-TOF MS provides an alternative approach to HBV genotyping.…”

Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory

“…Haplotyping can provide details regarding viral subpopulation complexity. This may be useful for antiviral resistance requiring multiple mutations (e.g., Entecavir), though they may predominantly colocalize to the same viral genome (27). Further study is needed to determine whether there is a clinical effect of differentiating patients with mutations present (that may or may not colocalize to the same viral genome) as tested by LiPA or Sanger sequencing compared to patients with mutations localized to a predominant haplotype (NGS).…”