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

PLoS One. 2013 Sep 18;8(9):e74249. doi: 10.1371/journal.pone.0074249. eCollection 2013.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Genome, Viral*
  • HIV-1 / classification
  • HIV-1 / genetics*
  • Haplotypes
  • High-Throughput Nucleotide Sequencing
  • Reassortant Viruses / genetics*
  • Recombination, Genetic*
  • Reverse Transcriptase Polymerase Chain Reaction

Grant support

This work was supported by the Swiss National Science Foundation [grants number 324700–120793 and 310030-141067 to HFG and KJM and CR32I2-127017 and CR32I2-146331 to NB, HFG and KJM and by the clinical research priority program viral infectious diseases, Zurich Primary HIV-infection Study by the university of Zurich (to HFG)]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.