title: Comparison of illumina and 454 deep sequencing in participants failing raltegravir-based antiretroviral therapy.
abstract: BACKGROUND
  The impact of raltegravir-resistant HIV-1 minority variants (MVs) on raltegravir treatment failure is unknown. Illumina sequencing offers greater throughput than 454, but sequence analysis tools for viral sequencing are needed. We evaluated Illumina and 454 for the detection of HIV-1 raltegravir-resistant MVs.
  
  
  METHODS
  A5262 was a single-arm study of raltegravir and darunavir/ritonavir in treatment-naïve patients. Pre-treatment plasma was obtained from 5 participants with raltegravir resistance at the time of virologic failure. A control library was created by pooling integrase clones at predefined proportions. Multiplexed sequencing was performed with Illumina and 454 platforms at comparable costs. Illumina sequence analysis was performed with the novel snp-assess tool and 454 sequencing was analyzed with V-Phaser.
  
  
  RESULTS
  Illumina sequencing resulted in significantly higher sequence coverage and a 0.095% limit of detection. Illumina accurately detected all MVs in the control library at ≥0.5% and 7/10 MVs expected at 0.1%. 454 sequencing failed to detect any MVs at 0.1% with 5 false positive calls. For MVs detected in the patient samples by both 454 and Illumina, the correlation in the detected variant frequencies was high (R2 = 0.92, P<0.001). Illumina sequencing detected 2.4-fold greater nucleotide MVs and 2.9-fold greater amino acid MVs compared to 454. The only raltegravir-resistant MV detected was an E138K mutation in one participant by Illumina sequencing, but not by 454.
  
  
  CONCLUSIONS
  In participants of A5262 with raltegravir resistance at virologic failure, baseline raltegravir-resistant MVs were rarely detected. At comparable costs to 454 sequencing, Illumina demonstrated greater depth of coverage, increased sensitivity for detecting HIV MVs, and fewer false positive variant calls.
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Jonathan Z
authors.initials: JZ
authors.lastname: Li
authors.affiliation: Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Brad
authors.initials: B
authors.lastname: Chapman
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Patrick
authors.initials: P
authors.lastname: Charlebois
authors.affiliation: Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Oliver
authors.initials: O
authors.lastname: Hofmann
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Brian
authors.initials: B
authors.lastname: Weiner
authors.affiliation: Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Alyssa J
authors.initials: AJ
authors.lastname: Porter
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America; Department of Virology, University of KwaZulu-Natal, National Health Laboratory Service, Durban, South Africa.
authors.forename: Reshmi
authors.initials: R
authors.lastname: Samuel
authors.affiliation: Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Saran
authors.initials: S
authors.lastname: Vardhanabhuti
authors.affiliation: Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Lu
authors.initials: L
authors.lastname: Zheng
authors.affiliation: Division of Infectious Diseases, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Joseph
authors.initials: J
authors.lastname: Eron
authors.affiliation: Division of Infectious Diseases, Northwestern University, Chicago, Illinois, United States of America.
authors.forename: Babafemi
authors.initials: B
authors.lastname: Taiwo
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Zody
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Henn
authors.affiliation: Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Kuritzkes
authors.affiliation: Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Winston
authors.initials: W
authors.lastname: Hide
authors.affiliation: University of Colorado.
authors.forename: Cara C
authors.initials: CC
authors.lastname: Wilson
authors.affiliation: Northwestern University.
authors.forename: Baiba I
authors.initials: BI
authors.lastname: Berzins
authors.affiliation: University of Alabama at Birmingham.
authors.forename: Edward P
authors.initials: EP
authors.lastname: Acosta
authors.affiliation: ACTG Operations Center, Social & Scientific Systems, Inc.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bastow
authors.affiliation: National Institute of Allergy and Infectious Diseases.
authors.forename: Peter S
authors.initials: PS
authors.lastname: Kim
authors.affiliation: National Institute of Allergy and Infectious Diseases.
authors.forename: Sarah W
authors.initials: SW
authors.lastname: Read
authors.affiliation: Frontier Science & Technology Research Foundation.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Janik
authors.affiliation: National Institute of Allergy and Infectious Diseases.
authors.forename: Debra S
authors.initials: DS
authors.lastname: Meres
authors.affiliation: Case Western Reserve University.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Lederman
authors.affiliation: Ohio State University.
authors.forename: Lori
authors.initials: L
authors.lastname: Mong-Kryspin
authors.affiliation: Henry Ford Hospital CRS.
authors.forename: Karl E
authors.initials: KE
authors.lastname: Shaw
authors.affiliation: University of Colorado CRS.
authors.forename: Louis G
authors.initials: LG
authors.lastname: Zimmerman
authors.affiliation: Merck.
authors.forename: Randi
authors.initials: R
authors.lastname: Leavitt
authors.affiliation: Tibotec.
authors.forename: Guy
authors.initials: G
authors.lastname: De La Rosa
authors.affiliation: ACTG Operations Center.
authors.forename: Amy
authors.initials: A
authors.lastname: Jennings
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D024882:Drug Resistance, Viral / Q000235:genetics; D015658:HIV Infections / Q000188:drug therapy* / Q000821:virology; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics* / Q000502:physiology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D011760:Pyrrolidinones / Q000494:pharmacology* / Q000627:therapeutic use; D000068898:Raltegravir Potassium; D017211:Treatment Failure
chemical_list: D019380:Anti-HIV Agents; D011760:Pyrrolidinones; D000068898:Raltegravir Potassium