A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.
A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.
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DOI：
10.1186/s12985-015-0280-x
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发表时间：
2015-04-09
期刊：
影响因子：
4.8
通讯作者：
Mak J
中科院分区：
文献类型：
--
作者：
Waugh C;Cromer D;Grimm A;Chopra A;Mallal S;Davenport M;Mak J
大规模的平行测序是一种有效的工具，通过揭示不同条件下细胞RNA谱的动态来剖析生物过程的调控。类似地，大规模平行测序可用于揭示经常在RNA病毒感染的宿主中发现的病毒准种的复杂性。然而，用于下一代测序（NGS）的cDNA文库的产生需要将RNA逆转录成cDNA并使用PCR扩增cDNA模板，这可能以幻影核酸物质的形式引入伪影，其可能使原始RNA谱的组成和解释发生偏差。使用HIV作为模型，我们已经表征了病毒RNA转化为cDNA过程中的主要错误来源，即过量的RNA模板和聚合酶（逆转录酶）的RNaseH活性。此外，我们还分析了PCR循环对重组体检测的影响，并评估了在我们的对照病毒生产过程中，高度相似的质粒DNA转染对重组体形成的贡献。我们已经确定了RNA模板浓度，RNA酶H活性的逆转录酶，和PCR条件的关键参数，必须仔细优化，以尽量减少嵌合的假象。使用我们优化的RT-PCR条件，结合我们改进的PCR扩增程序，我们开发了一种可靠的技术，用于使用NGS技术准确测定RNA种类。
Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles. Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses. We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts. Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.
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影响因子：
3.7
作者：
Di Giallonardo F;Zagordi O;Duport Y;Leemann C;Joos B;Künzli-Gontarczyk M;Bruggmann R;Beerenwinkel N;Günthard HF;Metzner KJ
通讯作者：
Metzner KJ
影响因子：
4.3
作者：
Schlub TE;Smyth RP;Grimm AJ;Mak J;Davenport MP
通讯作者：
Davenport MP
DOI：
10.1073/pnas.0306764101
发表时间：
2004-03-23
影响因子：
11.1
作者：
Levy, DN;Aldrovandi, GM;Shaw, GM
通讯作者：
Shaw, GM
DOI：
10.1073/pnas.221289898
发表时间：
2001-10-09
影响因子：
11.1
作者：
Hwang, CK;Svarovskaia, ES;Pathak, VK
通讯作者：
Pathak, VK
影响因子：
9.4
作者：
Avidor, Boaz;Girshengorn, Shirley;Turner, Dan
通讯作者：
Turner, Dan