Genome-Tagged Amplification (GTA): a PCR-based method to prepare sample-tagged amplicons from hundreds of individuals for next generation sequencing
Genome-Tagged Amplification (GTA): a PCR-based method to prepare sample-tagged amplicons from hundreds of individuals for next generation sequencing
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DOI：
10.1007/s11032-014-0090-7
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发表时间：
2014-10-01
影响因子：
3.1
通讯作者：
Syed, Naeem
中科院分区：
文献类型：
--
作者：
Ho, Thien;Cardle, Linda;Syed, Naeem
在大量个体中对基因组的相对小部分的序列进行采样是群体遗传学和关联遗传学方法的重要目标。然而，目前可用的“序列捕获”方法要么需要昂贵的仪器，要么在处理高样本数和相对小的靶尺寸时存在问题。我们开发了基因组标记扩增（GTA）作为一种灵活的基于PCR的方法，用于从数百个样品中制备数百个扩增子的池，用于下一代测序。该方法涉及在限制性位点用条形码衔接子标记基因组DNA，然后从侧翼DNA进行PCR扩增。它可以自由扩展样品数量和扩增子数量，并且没有专门的设备要求。提供了一种优化方案，其提供了96 x 192个样本x扩增子组合的矩阵，对应于完整的454钛运行。最初，我们使用454测序;然而，GTA可以容易地适应Illumina测序平台，因为在该系统中读取长度显著增加。
Sampling the sequence of a relatively small fraction of the genome in large numbers of individuals is an important objective for population genetics and association genetics approaches. However, currently available 'sequence capture' methods either require expensive instrumentation or have problems dealing with high sample numbers and relatively small target sizes. We have developed Genome-Tagged Amplification (GTA) as a flexible PCR-based method for preparing pools of hundreds of amplicons from hundreds of samples for next generation sequencing. The method involves tagging of genomic DNA with barcode adapters at restriction sites, followed by PCR amplification from flanking DNA. It is freely scalable for both sample number and amplicon number and has no specialized equipment requirement. An optimized protocol is presented which provides a matrix of 96 x 192 combinations of samples x amplicons, corresponding to a complete 454 Titanium run. Initially, we used 454 sequencing; however, GTA could easily be adapted to Illumina sequencing platforms as read lengths have significantly increased in this system.