Comparison of Illumina and 454 Deep Sequencing in Participants Failing Raltegravir-Based Antiretroviral Therapy

BackgroundThe 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.MethodsA5262 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.ResultsIllumina 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, PConclusionsIn 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.

研究背景：拉替拉韦耐药型HIV-1次要变异体（minority variants，MVs）对拉替拉韦治疗失败的影响目前尚不明确。Illumina测序（Illumina）的通量高于454测序（454），但目前仍亟需适配病毒测序的序列分析工具。本研究针对Illumina与454两种测序平台在检测HIV-1拉替拉韦耐药型次要变异体中的应用效果进行了评估。
研究方法：A5262是一项针对初治患者采用拉替拉韦与达芦那韦/利托那韦联合治疗的单臂临床试验。本研究从5例在病毒学失败时检出拉替拉韦耐药的受试者中获取了治疗前血浆样本。通过按预设比例混合整合酶克隆构建了对照文库。以相当的成本分别采用Illumina与454平台完成了多重测序。Illumina测序数据采用新型snp-assess工具进行分析，454测序数据则通过V-Phaser工具完成分析。
研究结果：Illumina测序展现出显著更高的序列覆盖度，且检测限为0.095%。在对照文库中，Illumina可准确检出≥0.5%比例的所有次要变异体，对于0.1%比例的10种预期变异体则成功检出7种。454测序无法检出0.1%比例的变异体，且出现5次假阳性调用。对于两种平台均检出的受试者样本中的次要变异体，二者检测到的变异体频率相关性极高（R²=0.92，P
研究结论：在A5262研究中，病毒学失败时检出拉替拉韦耐药的受试者里，基线水平的拉替拉韦耐药型次要变异体检出率极低。在与454测序成本相当的前提下，Illumina测序展现出更高的覆盖深度、更强的HIV次要变异体检测灵敏度，且假阳性变异体调用次数更少。