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Front. Plant Sci. | doi: 10.3389/fpls.2018.00201

Multiplex PCR Targeted Amplicon Sequencing (MTA-seq): Simple, Flexible, and Versatile SNP Genotyping by Highly Multiplexed PCR Amplicon Sequencing

 Yoshihiko Onda1, 2,  Kotaro Takahagi1, 2, 3, Minami Shimizu1,  Komaki Inoue1 and  Keiichi Mochida1, 2, 4, 5*
  • 1Center for Sustainable Resource Science, RIKEN, Japan
  • 2Kihara Institute for Biological Research, Yokohama City University, Japan
  • 3Graduate School of Nanobioscience, Yokohama City University, Japan
  • 4RIKEN Center for Sustainable Resource Science (CSRS), Japan
  • 5Institute of Plant Science and Resource, Okayama University, Japan

Next-generation sequencing technologies have enabled genome re-sequencing for exploring genome-wide polymorphisms among individuals, as well as targeted re-sequencing for rapid and simultaneous detection of polymorphisms in genes associated with various biological functions. Therefore, a simple and robust method for targeted re-sequencing should facilitate genotyping in a wide range of biological fields. In this study, we developed a simple, custom, targeted re-sequencing method, designated “multiplex PCR targeted amplicon sequencing (MTA-seq),” and applied it to genotyping of the model grass Brachypodium distachyon. To assess the practical usability of MTA-seq, we applied it to genotyping of genome-wide single-nucleotide polymorphisms (SNPs) identified in natural accessions (Bd1-1, Bd3-1, Bd21-3, Bd30-1, Koz-1, Koz-3, and Koz-4) by comparing the re-sequencing data to that of reference accession Bd21. Examination of SNP-genotyping accuracy in 443 amplicons from eight parental accessions and an F1 progeny derived by crossing of Bd21 and Bd3-1 revealed that ~95% of the SNPs were correctly called. The assessment suggested that the method provided an efficient framework for accurate and robust SNP genotyping. The method described here enables easy design of custom target SNP-marker panels in various organisms, facilitating a wide range of high-throughput genetic applications, such as genetic mapping, population analysis, molecular breeding, and genomic diagnostics.

Keywords: Marker panel, SNP, genotyping, Amplicon sequence, natural accession, Brachypodium distachyon

Received: 05 Jul 2017; Accepted: 02 Feb 2018.

Edited by:

Basil J. Nikolau, Iowa State University, United States

Reviewed by:

Sara Pinosio, Institute of Applied Genomics, Italy
Julia C. Meitz-Hopkins, Stellenbosch University, South Africa
Robert Henry, The University of Queensland, Australia  

Copyright: © 2018 Onda, Takahagi, Shimizu, Inoue and Mochida. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Keiichi Mochida, RIKEN, Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Wako, 230-0045, Kanagawa, Japan,