A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing.
A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing.
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DOI：
10.1371/journal.pone.0146687
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
454 HIV-1 Alpha Study Group
中科院分区：
文献类型：
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作者：
St John EP;Simen BB;Turenchalk GS;Braverman MS;Abbate I;Aerssens J;Bouchez O;Gabriel C;Izopet J;Meixenberger K;Di Giallonardo F;Schlapbach R;Paredes R;Sakwa J;Schmitz-Agheguian GG;Thielen A;Victor M;Metzner KJ;Däumer MP;454 HIV-1 Alpha Study Group
超深度测序不仅在研究中而且在诊断中的使用越来越多。为了在临床实验室中实施用于常规诊断的超深度测序测定，实验室内和实验室间测试是至关重要的。进行了一项多中心研究，以验证针对蛋白酶/逆转录酶（RTP）和env（V3）区域的454 Life Sciences GS FLX Titanium系统的更新检测设计，以识别HIV-1耐药突变并确定高灵敏度的共受体使用。该研究包括30份HIV-1亚型B和6份亚型非B样本，病毒滴度（VT）为3，940 - 447，400拷贝/mL，两个稀释系列（52，129 - 1，340和25，130 -734拷贝/mL）和一式三份样本。使用条形码化引物产生跨越PR密码子10-99、RT密码子1-251和整个V3区的扩增子。使用GS扩增子变体分析仪和geno 2 pheno进行向性分析。为了进行比较，使用ViroSeq HIV-1基因分型系统进行群体测序。RTP（IQR 592- 3，488）和V3（IQR 786- 3，695）的11个位点的中位数测序深度分别为每个位置1，829个读数和2，410个读数。10个预选的耐药变异体在各研究中心进行了测量，并在所有研究中心的数据中显示出高度的实验室间相关性（P<0.001）。质粒混合物的一式三份样品证实了实验室间的高度一致性（平均值% ±标准差：4.6 ±0.5、4.8 ±0.4、4.9 ±0.3），并显示了良好的实验室内一致性（平均值%范围±标准差范围：4.2-5.2 ± 0.04-0.65）。在两个稀释系列中，没有>20%的变体被遗漏，在大多数位点检测到2-10%的变体（即使在低VT下），并且在一些位点检测到1-2%的变体。通过群体测序检测的所有突变也通过UDS检测。该检测设计提供了一种准确且可重复的方法来分析HIV-1突变谱，即使变异频率远低于群体测序常规检测的频率。
Ultra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance. A multicenter study was conducted to validate an updated assay design for 454 Life Sciences’ GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity. The study included 30 HIV-1 subtype B and 6 subtype non-B samples with viral titers (VT) of 3,940–447,400 copies/mL, two dilution series (52,129–1,340 and 25,130–734 copies/mL), and triplicate samples. Amplicons spanning PR codons 10–99, RT codons 1–251 and the entire V3 region were generated using barcoded primers. Analysis was performed using the GS Amplicon Variant Analyzer and geno2pheno for tropism. For comparison, population sequencing was performed using the ViroSeq HIV-1 genotyping system. The median sequencing depth across the 11 sites was 1,829 reads per position for RTP (IQR 592–3,488) and 2,410 for V3 (IQR 786–3,695). 10 preselected drug resistant variants were measured across sites and showed high inter-laboratory correlation across all sites with data (P<0.001). The triplicate samples of a plasmid mixture confirmed the high inter-laboratory consistency (mean% ± stdev: 4.6 ±0.5, 4.8 ±0.4, 4.9 ±0.3) and revealed good intra-laboratory consistency (mean% range ± stdev range: 4.2–5.2 ± 0.04–0.65). In the two dilutions series, no variants >20% were missed, variants 2–10% were detected at most sites (even at low VT), and variants 1–2% were detected by some sites. All mutations detected by population sequencing were also detected by UDS. This assay design results in an accurate and reproducible approach to analyze HIV-1 mutant spectra, even at variant frequencies well below those routinely detectable by population sequencing.