Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination.

Di Giallonardo, Francesca; Zagordi, Osvaldo; Duport, Yannick; Leemann, Christine; Joos, Beda; Künzli-Gontarczyk, Marzanna; Bruggmann, Rémy; Beerenwinkel, Niko; Günthard, Huldrych F.; Metzner, Karin J. (2013). Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination. PLoS ONE, 8(9), e74249. Public Library of Science 10.1371/journal.pone.0074249

Preview

Text journal.pone.0074249.pdf - Published Version Available under License Creative Commons: Attribution (CC-BY). Download (1MB) | Preview

Next-generation sequencing (NGS) is a valuable tool for the detection and quantification of HIV-1 variants in vivo. However, these technologies require detailed characterization and control of artificially induced errors to be applicable for accurate haplotype reconstruction. To investigate the occurrence of substitutions, insertions, and deletions at the individual steps of RT-PCR and NGS, 454 pyrosequencing was performed on amplified and non-amplified HIV-1 genomes. Artificial recombination was explored by mixing five different HIV-1 clonal strains (5-virus-mix) and applying different RT-PCR conditions followed by 454 pyrosequencing. Error rates ranged from 0.04-0.66% and were similar in amplified and non-amplified samples. Discrepancies were observed between forward and reverse reads, indicating that most errors were introduced during the pyrosequencing step. Using the 5-virus-mix, non-optimized, standard RT-PCR conditions introduced artificial recombinants in a fraction of at least 30% of the reads that subsequently led to an underestimation of true haplotype frequencies. We minimized the fraction of recombinants down to 0.9-2.6% by optimized, artifact-reducing RT-PCR conditions. This approach enabled correct haplotype reconstruction and frequency estimations consistent with reference data obtained by single genome amplification. RT-PCR conditions are crucial for correct frequency estimation and analysis of haplotypes in heterogeneous virus populations. We developed an RT-PCR procedure to generate NGS data useful for reliable haplotype reconstruction and quantification.

Interest & Impact

Downloads

212 since deposited on 24 Jul 2014
12 in the past 12 months

Citations

5 Citations in Web of Science ®
6 Citations in Scopus

Search

in Google Scholar™

Services

Actions (login required)

Edit item

Actions (login required)

Edit item