Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination.
Next-generation sequencing of HIV-1 RNA genomes: determination of error rates and minimizing artificial recombination.
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DOI：
10.1371/journal.pone.0074249
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Metzner KJ
中科院分区：
文献类型：
--
作者：
Di Giallonardo F;Zagordi O;Duport Y;Leemann C;Joos B;Künzli-Gontarczyk M;Bruggmann R;Beerenwinkel N;Günthard HF;Metzner KJ
下一代测序（NGS）是一种有价值的工具，用于检测和定量体内HIV-1变异体。然而，这些技术需要详细的表征和人工诱导的错误的控制，以适用于准确的单倍型重建。为了研究在RT-PCR和NGS的各个步骤中置换、插入和缺失的发生，对扩增和未扩增的HIV-1基因组进行了454焦磷酸测序。通过混合五种不同的HIV-1克隆株（5种病毒混合物）并应用不同的RT-PCR条件，然后进行454焦磷酸测序来探索人工重组。错误率范围为0.04-0.66%，扩增和非扩增样本相似。在正向和反向读取之间观察到不一致性，表明大多数错误是在焦磷酸测序步骤期间引入的。使用5种病毒混合物，非优化的标准RT-PCR条件在至少30%的读段中引入人工重组体，这随后导致低估真实单倍型频率。我们通过优化的、减少人为因素的RT-PCR条件，将重组子的分数降至0.9-2.6%。这种方法能够实现正确的单倍型重建和频率估计与通过单基因组扩增获得的参考数据一致。RT-PCR条件对于异质性病毒群体中单倍型的正确频率估计和分析至关重要。我们开发了一种RT-PCR程序，以产生可靠的单倍型重建和定量有用的NGS数据。
Next-generation sequencing (NGS) is a valuable tool for the detection and quantification of HIV-1 variants in vivo. However, these technologies require detailed characterization and control of artificially induced errors to be applicable for accurate haplotype reconstruction. To investigate the occurrence of substitutions, insertions, and deletions at the individual steps of RT-PCR and NGS, 454 pyrosequencing was performed on amplified and non-amplified HIV-1 genomes. Artificial recombination was explored by mixing five different HIV-1 clonal strains (5-virus-mix) and applying different RT-PCR conditions followed by 454 pyrosequencing. Error rates ranged from 0.04–0.66% and were similar in amplified and non-amplified samples. Discrepancies were observed between forward and reverse reads, indicating that most errors were introduced during the pyrosequencing step. Using the 5-virus-mix, non-optimized, standard RT-PCR conditions introduced artificial recombinants in a fraction of at least 30% of the reads that subsequently led to an underestimation of true haplotype frequencies. We minimized the fraction of recombinants down to 0.9–2.6% by optimized, artifact-reducing RT-PCR conditions. This approach enabled correct haplotype reconstruction and frequency estimations consistent with reference data obtained by single genome amplification. RT-PCR conditions are crucial for correct frequency estimation and analysis of haplotypes in heterogeneous virus populations. We developed an RT-PCR procedure to generate NGS data useful for reliable haplotype reconstruction and quantification.
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影响因子：
12.3
作者：
Huse SM;Huber JA;Morrison HG;Sogin ML;Welch DM
通讯作者：
Welch DM
DOI：
10.1093/cid/ciq164
发表时间：
2011-02-15
期刊：
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
影响因子：
--
作者：
Kouyos RD;von Wyl V;Yerly S;Böni J;Rieder P;Joos B;Taffé P;Shah C;Bürgisser P;Klimkait T;Weber R;Hirschel B;Cavassini M;Rauch A;Battegay M;Vernazza PL;Bernasconi E;Ledergerber B;Bonhoeffer S;Günthard HF;Swiss HIV Cohort Study
通讯作者：
Swiss HIV Cohort Study
含利托那韦的蛋白酶抑制剂增强型抗逆转录病毒疗法能有效抑制初次感染 HIV-1 时出现的少数耐药 HIV-1 型 (HIV-1) 变异株
影响因子：
6.4
作者：
Metzner, Karin J.;Rauch, Pia;Guenthard, Huldrych F.
通讯作者：
Guenthard, Huldrych F.
DOI：
10.1093/bioinformatics/btq365
发表时间：
2010-09-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Balzer S;Malde K;Lanzén A;Sharma A;Jonassen I
通讯作者：
Jonassen I
影响因子：
5.8
作者：
Maydt, J;Lengauer, T
通讯作者：
Lengauer, T