通过扩增发夹连接的通用靶标进行靶标富集,用于下一代测序分析。
Target-enrichment through amplification of hairpin-ligated universal targets for next-generation sequencing analysis.
作者信息
Singh Pallavi, Nayak Rajesh, Kwon Young Min
机构信息
Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, USA.
出版信息
Methods Mol Biol. 2011;733:267-78. doi: 10.1007/978-1-61779-089-8_19.
With rapid development of next-generation sequencing (NGS) technologies, it is becoming increasingly feasible to sequence entire genomes of various organisms from virus to human. However, in many occasions, it is still more practical to sequence and analyze only small regions of the entire genome that are informative for the purpose of the experiment. Although many target-enrichment or target capture methods exist, each method has its own strength and weakness in terms of the number of enriched targets, specificity, drop-off rate, and uniformity in capturing target DNA sequences. Many applications require a consistently low drop-off rate and high uniformity of enriched targets for routine collection of meaningful data. Here, we describe a simple and robust PCR-based protocol that can allow simultaneous amplification of numerous target regions. This method employs target-specific hairpin selectors to create DNA templates that contain target regions flanked by common universal priming sequences. We demonstrated the utility of this method by applying it for simultaneous amplification of 21 targets in the range of 191-604 bp from 41 different Salmonella strains using bar-coded universal primers. Analysis of 454 FLX pyrosequencing data demonstrated the promising performance of this method in terms of specificity and uniformity. This method, with great potential for robust amplification of hundreds of targets, should find broad applications for efficient analysis of multiple genomic targets for various experimental goals.
随着下一代测序(NGS)技术的迅速发展,对从病毒到人类等各种生物体的全基因组进行测序变得越来越可行。然而,在许多情况下,仅对全基因组中对实验目的有信息价值的小区域进行测序和分析仍然更为实际。尽管存在许多靶标富集或靶标捕获方法,但每种方法在富集靶标的数量、特异性、脱落率以及捕获靶标DNA序列的均匀性方面都有其自身的优缺点。许多应用需要始终保持较低的脱落率和较高的富集靶标均匀性,以便常规收集有意义的数据。在此,我们描述了一种基于PCR的简单且稳健的方案,该方案可同时扩增众多靶标区域。此方法采用靶标特异性发夹选择器来创建DNA模板,这些模板包含由通用引物序列侧翼的靶标区域。我们通过使用条形码通用引物对来自41种不同沙门氏菌菌株的191 - 604 bp范围内的21个靶标进行同时扩增,证明了该方法的实用性。对454 FLX焦磷酸测序数据的分析表明,该方法在特异性和均匀性方面具有良好的性能。这种方法具有强大的潜力,可对数百个靶标进行稳健扩增,应该会在针对各种实验目标高效分析多个基因组靶标方面找到广泛的应用。