%0 Journal Article %1 zagordi2010error %A Zagordi, O %A Klein, R %A Däumer, M %A Beerenwinkel, N %D 2010 %J Nucleic Acids Res %K HIV haplotype_inference methods quasispecies software %N 21 %P 7400-7409 %R 10.1093/nar/gkq655 %T Error correction of next-generation sequencing data and reliable estimation of HIV quasispecies %U http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2995073/?tool=pmcentrez %V 38 %X 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.

@article{zagordi2010error, 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.}, added-at = {2012-04-09T06:23:04.000+0200}, author = {Zagordi, O and Klein, R and D{\"a}umer, M and Beerenwinkel, N}, biburl = {https://www.bibsonomy.org/bibtex/2de41a33a418b9e7515d717cad14dc6ed/peter.ralph}, description = {Describes ShoRAH.}, doi = {10.1093/nar/gkq655}, interhash = {7fcb4d826e7f1edee3cd0b7a17989e23}, intrahash = {de41a33a418b9e7515d717cad14dc6ed}, journal = {Nucleic Acids Res}, keywords = {HIV haplotype_inference methods quasispecies software}, month = nov, number = 21, pages = {7400-7409}, pmid = {20671025}, timestamp = {2012-04-09T06:23:04.000+0200}, title = {Error correction of next-generation sequencing data and reliable estimation of {HIV} quasispecies}, url = {http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2995073/?tool=pmcentrez}, volume = 38, year = 2010 }