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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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 × 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.
KeywordsNext generation sequencing Barley PCR Pooling
The authors thank Patrick Schnable for suggestions on barcoding, Dan Ashlock for generating and providing barcodes for this study and Jeffrey Jeddeloh for communicating unpublished observations on hybridization capture technologies. We also thank Margaret Hughes, Garry Cusack and the Roche-454 Delaware sequencing facility for much help and advice with 454 sequencing. This work was supported by Biotechnology and Biological Sciences Research Council grant BB/E003184/1.
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