title: Identification of minority resistance mutations in the HIV-1 integrase coding region using next generation sequencing.
abstract: BACKGROUND
  The current widely applied standard method to screen for HIV-1 genotypic resistance is based on Sanger population sequencing (Sseq), which does not allow for the identification of minority variants (MVs) below the limit of detection for the Sseq-method in patients receiving integrase strand-transfer inhibitors (INSTI). Next generation sequencing (NGS) has facilitated the detection of MVs at a much deeper level than Sseq.
  
  
  OBJECTIVE
  Here, we compared Illumina MiSeq and Sseq approaches to evaluate the detection of MVs involved in resistance to the three commonly used INSTI: raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG).
  
  
  METHODS
  NGS and Sseq were used to analyze RT-PCR products of the HIV-1 integrase coding region from six patients and in serial samples from two patients. NGS sequences were assembled and analyzed using the low frequency variant detection (LFVDT) tool in CLC genomic workbench.
  
  
  RESULTS
  Sseq detected INSTI resistance and accessory mutations in three of the patients (called INSTI Res+), while no resistance or accessory mutations were detected in the remaining three patients (called INSTI Res-). Additional INSTI resistance and/or accessory mutations were detected by NGS analysis of integrase sequences from all three INSTI Res+ and one INSTI Res- patient.
  
  
  CONCLUSIONS
  Our observations suggested that NGS demonstrated a higher sensitivity than sSEQ in the identification of INSTI relevant MVs both in patients at treatment baseline and in patients receiving INSTI therapy. Thus NGS can be a valuable tool in monitoring of antiretroviral minority resistance in patients receiving INSTI therapy.
authors.affiliation: Department of Microbiological Diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark. Electronic address: FON@ssi.dk.
authors.forename: Jannik
authors.initials: J
authors.lastname: Fonager
authors.affiliation: Department of Microbiology and Infection Control, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Jonas T
authors.initials: JT
authors.lastname: Larsson
authors.affiliation: Department of Microbiological Diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Hussing
authors.affiliation: Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark.
authors.forename: Frederik
authors.initials: F
authors.lastname: Neess Engsig
authors.affiliation: Department of Microbiological Diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Nielsen
authors.affiliation: Department of Microbiological Diagnostics and Virology, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Thea Kølsen
authors.initials: TK
authors.lastname: Fischer
mesh_terms: D000293:Adolescent; D002675:Child, Preschool; D024882:Drug Resistance, Viral; D019427:HIV Integrase / Q000235:genetics*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006575:Heterocyclic Compounds, 3-Ring; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D010078:Oxazines; D010879:Piperazines; D011728:Pyridones; D015363:Quinolones; D000068898:Raltegravir Potassium; D017423:Sequence Analysis, RNA / Q000379:methods*; D000099226:Dolutegravir
chemical_list: D019427:HIV Integrase; D006575:Heterocyclic Compounds, 3-Ring; D010078:Oxazines; D010879:Piperazines; D011728:Pyridones; D015363:Quinolones; D000068898:Raltegravir Potassium; D000099226:Dolutegravir; C509700:elvitegravir
keywords: Deep sequencing; Hiv-1; Illumina MiSeq; Integrase; Minority variants; Resistance