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PLoS One DOI:10.1371/journal.pone.0090485

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

Publication TypeJournal Article
Year of Publication2014
AuthorsLi, JZ, Chapman, B, Charlebois, P, Hofmann, O, Weiner, B, Porter, AJ, Samuel, R, Vardhanabhuti, S, Zheng, L, Eron, J, Taiwo, B, Zody, MC, Henn, MR, Kuritzkes, DR, Hide, W, Wilson, CC, Berzins, BI, Acosta, EP, Bastow, B, Kim, PS, Read, SW, Janik, J, Meres, DS, Lederman, MM, Mong-Kryspin, L, Shaw, KE, Zimmerman, LG, Leavitt, R, De La Rosa, G, Jennings, A
Corporate AuthorsACTG A5262 Study Team
JournalPLoS One
Date Published2014
KeywordsAnti-HIV Agents, Drug Resistance, Viral, High-Throughput Nucleotide Sequencing, HIV Infections, HIV-1, Humans, Mutation, Pyrrolidinones, Raltegravir Potassium, Treatment Failure

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

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.


Alternate JournalPLoS ONE
PubMed ID24603872
PubMed Central IDPMC3946168
Grant ListUM1 AI069423 / AI / NIAID NIH HHS / United States
K08 AI100699 / AI / NIAID NIH HHS / United States
U01 AI068636 / AI / NIAID NIH HHS / United States
UM1 AI069471 / AI / NIAID NIH HHS / United States
UM1 AI069412 / AI / NIAID NIH HHS / United States
UM1 AI068634 / AI / NIAID NIH HHS / United States
U01 AI068634 / AI / NIAID NIH HHS / United States
UL1 TR000439 / TR / NCATS NIH HHS / United States
UM1 AI106701 / AI / NIAID NIH HHS / United States
UL1 RR025758 / RR / NCRR NIH HHS / United States
UL1 RR 025758 / RR / NCRR NIH HHS / United States
P30 AI060354 / AI / NIAID NIH HHS / United States
UM1 AI068636 / AI / NIAID NIH HHS / United States
P30 AI050410 / AI / NIAID NIH HHS / United States