Modular tagging of amplicons using a single PCR for high-throughput sequencing
Modular tagging of amplicons using a single PCR for high-throughput sequencing
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DOI：
10.1111/1755-0998.12162
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发表时间：
2014-01-01
影响因子：
7.7
通讯作者：
Cooper, Alan
中科院分区：
文献类型：
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作者：
Clarke, Laurence J.;Czechowski, Paul;Cooper, Alan
PCR扩增的高通量测序(HTS)正成为对一个或多个目标基因座进行系统发育和DNA条码研究的首选方法。尽管HTS的发展使得快速产生大量的DNA序列数据成为可能，但为HTS准备扩增仍然是一个限速步骤。例如，HTS平台要求在要测序的DNA片段的第5和第3端存在特定于平台的适配序列。此外，通常添加短多路复用识别符(MID)标签以允许在单个HTS运行中汇集多个样本。现有的将HTS接头和MID标签结合到PCR扩增中的方法要么效率低下，需要多个酶反应和清理步骤，要么应用于多个样本或基因座(融合引物)成本高昂。我们描述了一种利用单次聚合酶链式反应扩增靶基因并通过连接子序列添加HTS接头和MID标签的方法。我们通过为不同的昆虫分类群生成两个线粒体基因座(COI和16S)的参考序列数据来演示我们的方法。我们的方法提供了一种灵活、经济和高效的方法来为高温超导制备扩增产物。
High-throughput sequencing (HTS) of PCR amplicons is becoming the method of choice to sequence one or several targeted loci for phylogenetic and DNA barcoding studies. Although the development of HTS has allowed rapid generation of massive amounts of DNA sequence data, preparing amplicons for HTS remains a rate-limiting step. For example, HTS platforms require platform-specific adapter sequences to be present at the 5 and 3 end of the DNA fragment to be sequenced. In addition, short multiplex identifier (MID) tags are typically added to allow multiple samples to be pooled in a single HTS run. Existing methods to incorporate HTS adapters and MID tags into PCR amplicons are either inefficient, requiring multiple enzymatic reactions and clean-up steps, or costly when applied to multiple samples or loci (fusion primers). We describe a method to amplify a target locus and add HTS adapters and MID tags via a linker sequence using a single PCR. We demonstrate our approach by generating reference sequence data for two mitochondrial loci (COI and 16S) for a diverse suite of insect taxa. Our approach provides a flexible, cost-effective and efficient method to prepare amplicons for HTS.