title: Error correction of next-generation sequencing data and reliable estimation of HIV quasispecies.
abstract: Next-generation sequencing technologies can be used to analyse genetically heterogeneous samples at unprecedented detail. The high coverage achievable with these methods enables the detection of many low-frequency variants. However, sequencing errors complicate the analysis of mixed populations and result in inflated estimates of genetic diversity. We developed a probabilistic Bayesian approach to minimize the effect of errors on the detection of minority variants. We applied it to pyrosequencing data obtained from a 1.5-kb-fragment of the HIV-1 gag/pol gene in two control and two clinical samples. The effect of PCR amplification was analysed. Error correction resulted in a two- and five-fold decrease of the pyrosequencing base substitution rate, from 0.05% to 0.03% and from 0.25% to 0.05% in the non-PCR and PCR-amplified samples, respectively. We were able to detect viral clones as rare as 0.1% with perfect sequence reconstruction. Probabilistic haplotype inference outperforms the counting-based calling method in both precision and recall. Genetic diversity observed within and between two clinical samples resulted in various patterns of phenotypic drug resistance and suggests a close epidemiological link. We conclude that pyrosequencing can be used to investigate genetically diverse samples with high accuracy if technical errors are properly treated.
authors.affiliation: Department of Biosystems Sciences and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Germany. osvaldo.zagordi@bsse.ethz.ch
authors.forename: Osvaldo
authors.initials: O
authors.lastname: Zagordi
authors.forename: Rolf
authors.initials: R
authors.lastname: Klein
authors.forename: Martin
authors.initials: M
authors.lastname: Däumer
authors.forename: Niko
authors.initials: N
authors.lastname: Beerenwinkel
mesh_terms: D000465:Algorithms; D016477:Artifacts; D001499:Bayes Theorem; D024882:Drug Resistance, Viral / Q000235:genetics; D005787:Gene Frequency; D015658:HIV Infections / Q000821:virology; D016333:HIV Protease / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1