Next-generation sequencing-based analysis of reverse transcriptase fidelity
Next-generation sequencing-based analysis of reverse transcriptase fidelity
复制标题
DOI：
10.1016/j.bbrc.2017.07.169
复制
发表时间：
2017-10-14
影响因子：
3.1
通讯作者：
Fujiwara, Shinsuke
中科院分区：
文献类型：
--
作者：
Yasukawa, Kiyoshi;Iida, Kei;Fujiwara, Shinsuke
在这项研究中，我们设计了一个简单而快速的方法来分析保真度的逆转录酶（RT）使用下一代测序（NGS）。该方法包括从标准RNA与含有14个随机碱基的标签和待测RT的引物进行cDNA合成反应，使用高保真DNA聚合酶进行PCR，以及NGS。通过将每个读段的序列与参考序列进行比较，鉴定突变。基于具有相同标签的序列读数是否含有相同突变，可以将突变鉴定为由于cDNA合成、PCR或NGS引入的错误。用莫洛尼鼠白血病病毒（MMLV）RT热稳定变异体MM 4或最近开发的具有RT活性的B家族DNA聚合酶的16-元组变异体RTX（来自Thermococcus kodakarensis）合成cDNA的错误率为0.75-1.0 × 10（-4）个错误/碱基，而在与野生型人类免疫缺陷病毒I型（HIV-1）RT反应中，为2.6 × 10（-4）个错误/碱基。总的来说，我们的方法可以以高通量的方式精确地评估具有不同反应条件的各种RT的保真度，而无需使用昂贵的光学器件和麻烦的接头连接。(C)2017爱思唯尔公司All rights reserved.
In this study, we devised a simple and rapid method to analyze fidelity of reverse transcriptase (RT) using next-generation sequencing (NGS). The method comprises a cDNA synthesis reaction from standard RNA with a primer containing a tag of 14 randomized bases and the RT to be tested, PCR using high-fidelity DNA polymerase, and NGS. By comparing the sequence of each read with the reference sequence, mutations were identified. The mutation can be identified to be due to an error introduced by either cDNA synthesis, PCR, or NGS based on whether the sequence reads with the same tag contain the same mutation or not. The error rates in cDNA synthesis with Moloney murine leukemia virus (MMLV) RT thermostable variant MM4 or the recently developed 16-tuple variant of family B DNA polymerase with RT activity, RTX, from Thermococcus kodakarensis, were 0.75-1.0 x 10(-4) errors/base, while that in the reaction with the wild-type human immunodeficiency virus type I (HIV-1) RT was 2.6 x 10(-4) errors/base. Overall, our method could precisely evaluate the fidelity of various RTs with different reaction conditions in a high-throughput manner without the use of expensive optics and troublesome adaptor ligation. (C) 2017 Elsevier Inc. All rights reserved.