A study has shown ultra-deep sequencing can detect low-frequency hepatitis B virus (HBV) drug-resistant mutations in infected samples, including mutations undetected by standard direct PCR sequencing.
The study, published in the Journal of Infectious Disease, outlines the results of a collaborative study between 454 Life Sciences and Stanford University, which used the 454 Sequencing System to examine blood samples from nucleoside and nucleotide reverse-transcriptase inhibitor (NRTI) treated and untreated individuals.
The study provides an insight into the dynamics of early stage NRTI resistance in HBV, as current methods limit detection to mutations only after they have become prevalent.
Hepatitis B virus is a chronic disease that causes severe inflammation of the liver.
Similar to HIV, HBV reverse-transcriptase has a high rate of replication, which generates a growing quantity of potentially drug-resistant strains within a host.
Current methods for characterising HBV sequence variability, such as direct PCR sequencing or population-based sequencing, detect mutations present to only >20 per cent of the virus population.
By using unbiased, highly sensitive ultra-deep sequencing, the researchers at Stanford University, led by Dr Robert Shafer and Dr Severine Margeridon-Thermet, were able to detect a variety of drug-resistant mutations present in as little as one per cent of the population.
The study found HBV drug-resistant mutations in 10 samples from five NRTI-treated individuals that were not detected by PCR.
Ultra-deep sequencing showed that several samples were co-infected with A and G genotypes, when direct PCR sequencing had only detected genotype G.
Dr Robert Shafer, principal author and associate professor of infectious disease at Stanford University, said: 'This study demonstrates the power of ultra-deep sequencing to uncover previously obscured HBV drug resistance.
'The expanded perspective on emerging and latent HBV drug resistance provided by this method may make it possible to improve the strategic use of HBV drugs to treat this lifelong infection in the future.' Roche-company 454 Life Sciences develops and sells the 454 Sequencing System for ultra high-throughput DNA sequencing.
Specific applications include de novo sequencing and re-sequencing of genomes, metagenomics, RNA analysis and targeted sequencing of DNA regions of interest.
The 454 Sequencing System features simple, unbiased sample preparation and long and accurate sequence reads, including paired-end reads.
The technology of the 454 Sequencing System has enabled hundreds of peer-reviewed studies in diverse research fields, including cancer and infectious-disease research, drug discovery, marine biology, anthropology and palaeontology.
