Development of a low bias method for characterizing viral populations using next generation sequencing technology.
Development of a low bias method for characterizing viral populations using next generation sequencing technology.
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DOI：
10.1371/journal.pone.0013564
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发表时间：
2010-10-22
期刊：
影响因子：
3.7
通讯作者：
Schaffer DV
中科院分区：
文献类型：
--
作者：
Willerth SM;Pedro HA;Pachter L;Humeau LM;Arkin AP;Schaffer DV
据估计，目前全世界有3800万人感染人类免疫缺陷病毒（HIV），每年还有410万人受到感染，因此了解这种病毒如何突变并产生耐药性以设计成功的治疗方法非常重要。我们报告了一种新的实验方法，用于扩增全长HIV基因组，而不使用序列特异性引物进行高通量DNA测序，然后从所得的大数据集组装全长病毒基因组序列。选择Illumina进行测序是因为与现有方法相比，它能够提供更大的HIV基因组覆盖率，从而能够更全面地表征所分析的HIV样品中存在的异质性。我们的新型扩增方法与Illumina测序相结合用于分析两种HIV群体：基于典型NL 4 -3毒株的同质HIV群体和从HIV患者感染的T细胞获得的异质病毒群体。此外，使用新的计算方法分析所得序列，以获得一致序列和几个多样性度量。这项研究展示了低偏差扩增方法如何与下一代DNA测序相结合，提供对艾滋病毒基因组的深入、完整的覆盖，从而能够更强有力地表征临床相关艾滋病毒群体中存在的准种，以及未来研究艾滋病毒如何响应选择压力而突变。
With an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies. We report a novel experimental method for amplifying full-length HIV genomes without the use of sequence-specific primers for high throughput DNA sequencing, followed by assembly of full length viral genome sequences from the resulting large dataset. Illumina was chosen for sequencing due to its ability to provide greater coverage of the HIV genome compared to prior methods, allowing for more comprehensive characterization of the heterogeneity present in the HIV samples analyzed. Our novel amplification method in combination with Illumina sequencing was used to analyze two HIV populations: a homogenous HIV population based on the canonical NL4-3 strain and a heterogeneous viral population obtained from a HIV patient's infected T cells. In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity. This study demonstrates how a lower bias amplification method in combination with next generation DNA sequencing provides in-depth, complete coverage of the HIV genome, enabling a stronger characterization of the quasispecies present in a clinically relevant HIV population as well as future study of how HIV mutates in response to a selective pressure.
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影响因子：
30.3
作者：
Leonard JN;Shah PS;Burnett JC;Schaffer DV
通讯作者：
Schaffer DV
影响因子：
14.9
作者：
Hoffmann C;Minkah N;Leipzig J;Wang G;Arens MQ;Tebas P;Bushman FD
通讯作者：
Bushman FD
未来研究植物病原体效应子的路线图。
影响因子：
4.9
作者：
Alfano, James R.
通讯作者：
Alfano, James R.
影响因子：
5.4
作者：
ADACHI, A;GENDELMAN, HE;MARTIN, MA
通讯作者：
MARTIN, MA
影响因子：
1.5
作者：
GIBBS, JS;REGIER, DA;DESROSIERS, RC
通讯作者：
DESROSIERS, RC