Genome-Tagged Amplification (GTA): a PCR-based method to prepare sample-tagged amplicons from hundreds of individuals for next generation sequencing

Thien Ho, Linda Cardle, Xin Xu, Micha Bayer, K. Silvas Jebakumar Prince, Raymond N. Mutava, David F. Marshall, Naeem Syed*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)977-988
JournalPlant Breeding
Volume34
Issue number3
DOIs
Publication statusPrint publication - Apr 2014
Externally publishedYes

Keywords

  • Next generation sequencing
  • Barley
  • PCR
  • Pooling

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