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PLoS One. 2014 Mar 6;9(3):e90485. doi: 10.1371/journal.pone.0090485. eCollection 2014.

Comparison of illumina and 454 deep sequencing in participants failing raltegravir-based antiretroviral therapy.

Author information

  • 1Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America.
  • 2Harvard School of Public Health, Boston, Massachusetts, United States of America.
  • 3Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
  • 4Division 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.
  • 5Division of Infectious Diseases, University of North Carolina, Chapel Hill, North Carolina, United States of America.
  • 6Division of Infectious Diseases, Northwestern University, Chicago, Illinois, United States of America.
  • 7University of Colorado.
  • 8Northwestern University.
  • 9University of Alabama at Birmingham.
  • 10ACTG Operations Center, Social & Scientific Systems, Inc.
  • 11National Institute of Allergy and Infectious Diseases.
  • 12Frontier Science & Technology Research Foundation.
  • 13Case Western Reserve University.
  • 14Ohio State University.
  • 15Henry Ford Hospital CRS.
  • 16University of Colorado CRS.
  • 17Merck.
  • 18Tibotec.
  • 19ACTG Operations Center.



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.


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.


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.


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.

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