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

Onda, Yoshihiko; Takahagi, Kotaro; Shimizu, Minami; Inoue, Komaki; Mochida, Keiichi

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

Journal Article · Fri Mar 23 04:00:00 EDT 2018 · Frontiers in Plant Science

DOI:https://doi.org/10.3389/fpls.2018.00201· OSTI ID:1628286

Onda, Yoshihiko ^[1]; Takahagi, Kotaro ^[2]; Shimizu, Minami ^[3]; Inoue, Komaki ^[3]; Mochida, Keiichi ^[4]

RIKEN Center for Sustainable Resource Science, Yokohama (Japan). Cellulose Production Research Team; Yokohama City Univ. (Japan). Kihara Inst. for Biological Research; DOE/OSTI
RIKEN Center for Sustainable Resource Science, Yokohama (Japan). Cellulose Production Research Team; Yokohama City Univ. (Japan). Kihara Inst. for Biological Research; Yokohama City Univ. (Japan). Inst. of Plant Science and Resource
RIKEN Center for Sustainable Resource Science, Yokohama (Japan). Cellulose Production Research Team
RIKEN Center for Sustainable Resource Science, Yokohama (Japan). Cellulose Production Research Team; Yokohama City Univ. (Japan). Kihara Inst. for Biological Research; Okayama Univ. (Japan). Inst. of Plance Science and Resource

Next-generation sequencing (NGS) technologies have enabled genome re-sequencing for exploring genome-wide polymorphisms among individuals, as well as targeted re-sequencing for the 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 the genotyping of the model grass Brachypodium distachyon. To assess the practical usability of MTA-seq, we applied it to the 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 with 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.

View Accepted Manuscript (DOE)

Research Organization:: RIKEN Center for Sustainable Resource Science, Yokohama (Japan)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: FG02-00ER41132

OSTI ID:: 1628286

Journal Information:: Frontiers in Plant Science, Journal Name: Frontiers in Plant Science Vol. 9; ISSN 1664-462X

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

References (63)

Comparison of a high-density genetic linkage map to genome features in the model grass Brachypodium distachyon Huo, Naxin; Garvin, David F.; You, Frank M. Theoretical and Applied Genetics, Vol. 123, Issue 3 https://doi.org/10.1007/s00122-011-1598-4	journal	May 2011
High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3 Vogel, John; Hill, Theresa Plant Cell Reports, Vol. 27, Issue 3 https://doi.org/10.1007/s00299-007-0472-y	journal	November 2007
Evaluation of a new NGS method based on a custom AmpliSeq library and Ion Torrent PGM sequencing for the fast detection of genetic variations in cardiomyopathies Millat, Gilles; Chanavat, Valérie; Rousson, Robert Clinica Chimica Acta, Vol. 433 https://doi.org/10.1016/j.cca.2014.03.032	journal	June 2014
Targeted next-generation sequencing (NGS) of nine candidate genes with custom AmpliSeq in patients and a cardiomyopathy risk group Glotov, Andrey S.; Kazakov, Sergey V.; Zhukova, Elena A. Clinica Chimica Acta, Vol. 446 https://doi.org/10.1016/j.cca.2015.04.014	journal	June 2015
Brachypodium distachyon genomics for sustainable food and fuel production Bevan, Michael W.; Garvin, David F.; Vogel, John P. Current Opinion in Biotechnology, Vol. 21, Issue 2 https://doi.org/10.1016/j.copbio.2010.03.006	journal	April 2010
Clinical Validation of a Next-Generation Sequencing Screen for Mutational Hotspots in 46 Cancer-Related Genes Singh, Rajesh R.; Patel, Keyur P.; Routbort, Mark J. The Journal of Molecular Diagnostics, Vol. 15, Issue 5 https://doi.org/10.1016/j.jmoldx.2013.05.003	journal	September 2013
Progress, challenges and the future of crop genomes Michael, Todd P.; VanBuren, Robert Current Opinion in Plant Biology, Vol. 24 https://doi.org/10.1016/j.pbi.2015.02.002	journal	April 2015
A framework for variation discovery and genotyping using next-generation DNA sequencing data DePristo, Mark A.; Banks, Eric; Poplin, Ryan Nature Genetics, Vol. 43, Issue 5 https://doi.org/10.1038/ng.806	journal	April 2011
Harnessing the power of RADseq for ecological and evolutionary genomics Andrews, Kimberly R.; Good, Jeffrey M.; Miller, Michael R. Nature Reviews Genetics, Vol. 17, Issue 2 https://doi.org/10.1038/nrg.2015.28	journal	January 2016
Genotype imputation for genome-wide association studies Marchini, Jonathan; Howie, Bryan Nature Reviews Genetics, Vol. 11, Issue 7 https://doi.org/10.1038/nrg2796	journal	June 2010
Genome-wide genetic marker discovery and genotyping using next-generation sequencing Davey, John W.; Hohenlohe, Paul A.; Etter, Paul D. Nature Reviews Genetics, Vol. 12, Issue 7 https://doi.org/10.1038/nrg3012	journal	June 2011
Ecological genomics of local adaptation Savolainen, Outi; Lascoux, Martin; Merilä, Juha Nature Reviews Genetics, Vol. 14, Issue 11 https://doi.org/10.1038/nrg3522	journal	October 2013
Using next-generation sequencing to isolate mutant genes from forward genetic screens Schneeberger, Korbinian Nature Reviews Genetics, Vol. 15, Issue 10 https://doi.org/10.1038/nrg3745	journal	August 2014
Population-level deficit of homozygosity unveils CPSF3 as an intellectual disability syndrome gene Arnadottir, Gudny A.; Oddsson, Asmundur; Jensson, Brynjar O. Nature Communications, Vol. 13, Issue 1 https://doi.org/10.1038/s41467-022-28330-8	journal	February 2022
Targeted A-to-G base editing of chloroplast DNA in plants Mok, Young Geun; Hong, Sunghyun; Bae, Su-Ji Nature Plants, Vol. 8, Issue 12 https://doi.org/10.1038/s41477-022-01279-8	journal	December 2022
CXCR4 signaling directs Igk recombination and the molecular mechanisms of late B lymphopoiesis Mandal, Malay; Okoreeh, Michael K.; Kennedy, Domenick E. Nature Immunology, Vol. 20, Issue 10 https://doi.org/10.1038/s41590-019-0468-0	journal	September 2019
VarScan: variant detection in massively parallel sequencing of individual and pooled samples Koboldt, Daniel C.; Chen, Ken; Wylie, Todd Bioinformatics, Vol. 25, Issue 17 https://doi.org/10.1093/bioinformatics/btp373	journal	June 2009
Brachypodium: a promising hub between model species and cereals Girin, Thomas; David, Laure C.; Chardin, Camille Journal of Experimental Botany, Vol. 65, Issue 19 https://doi.org/10.1093/jxb/eru376	journal	September 2014
Determination of growth stages and metabolic profiles in Brachypodium distachyon for comparison of developmental context with Triticeae crops Onda, Yoshihiko; Hashimoto, Kei; Yoshida, Takuhiro Proceedings of the Royal Society B: Biological Sciences, Vol. 282, Issue 1811 https://doi.org/10.1098/rspb.2015.0964	journal	July 2015
Brachypodium distachyon . A New Model System for Functional Genomics in Grasses Draper, John; Mur, Luis A. J.; Jenkins, Glyn Plant Physiology, Vol. 127, Issue 4 https://doi.org/10.1104/pp.010196	journal	December 2001
Exploiting the Brachypodium Tool Box in cereal and grass research Mur, Luis A. J.; Allainguillaume, Joel; Catalán, Pilar New Phytologist, Vol. 191, Issue 2 https://doi.org/10.1111/j.1469-8137.2011.03748.x	journal	May 2011
Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines Gordon, Sean P.; Priest, Henry; Des Marais, David L. The Plant Journal, Vol. 79, Issue 3 https://doi.org/10.1111/tpj.12569	journal	July 2014
Brachypodium distachyon as a Genetic Model System Kellogg, Elizabeth A. Annual Review of Genetics, Vol. 49, Issue 1 https://doi.org/10.1146/annurev-genet-112414-055135	journal	November 2015
Development of SSR markers and analysis of diversity in Turkish populations of Brachypodium distachyon Vogel, John P.; Tuna, Metin; Budak, Hikmet BMC Plant Biology, Vol. 9, Issue 1 https://doi.org/10.1186/1471-2229-9-88	journal	January 2009
Genome-wide association studies in plants: the missing heritability is in the field Brachi, Benjamin; Morris, Geoffrey P.; Borevitz, Justin O. Genome Biology, Vol. 12, Issue 10 https://doi.org/10.1186/gb-2011-12-10-232	journal	January 2011
Fine Mapping of the Bsr1 Barley Stripe Mosaic Virus Resistance Gene in the Model Grass Brachypodium distachyon Cui, Yu; Lee, Mi Yeon; Huo, Naxin PLoS ONE, Vol. 7, Issue 6 https://doi.org/10.1371/journal.pone.0038333	journal	June 2012
DNA Analysis of Herbarium Specimens of the Grass Weed Alopecurus myosuroides Reveals Herbicide Resistance Pre-Dated Herbicides Délye, Christophe; Deulvot, Chrystel; Chauvel, Bruno PLoS ONE, Vol. 8, Issue 10 https://doi.org/10.1371/journal.pone.0075117	journal	October 2013
Increasing Genome Sampling and Improving SNP Genotyping for Genotyping-by-Sequencing with New Combinations of Restriction Enzymes Fu, Yong-Bi; Peterson, Gregory W.; Dong, Yibo G3: Genes\|Genomes\|Genetics, Vol. 6, Issue 4 https://doi.org/10.1534/g3.115.025775	journal	January 2016
Development of Genetic and Genomic Research Resources for , a New Model System for Grass Crop Research Garvin, David F.; Gu, Yong-Qiang; Hasterok, Robert Crop Science, Vol. 48, Issue Supplement_1 https://doi.org/10.2135/cropsci2007.06.0332tpg	journal	January 2008
Exploring Genetic Diversity in Plants Using High-Throughput Sequencing Techniques Onda, Yoshihiko; Mochida, Keiichi Current Genomics, Vol. 17, Issue 4 https://doi.org/10.2174/1389202917666160331202742	journal	June 2016
Current knowledge in lentil genomics and its application for crop improvement Kumar, Shiv; Rajendran, Karthika; Kumar, Jitendra Frontiers in Plant Science, Vol. 6 https://doi.org/10.3389/fpls.2015.00078	journal	February 2015
Deciphering regulation in eukaryotic cell: from sequence to function Boeva, Valentina arXiv https://doi.org/10.48550/arxiv.1409.6661	preprint	January 2014
A novel three-round multiplex PCR for SNP genotyping with next generation sequencing Chen, Ke; Zhou, Yu-xun; Li, Kai Analytical and Bioanalytical Chemistry, Vol. 408, Issue 16 https://doi.org/10.1007/s00216-016-9536-6	journal	April 2016
Combining Highly Multiplexed PCR with Semiconductor-Based Sequencing for Rapid Cancer Genotyping Beadling, Carol; Neff, Tanaya L.; Heinrich, Michael C. The Journal of Molecular Diagnostics, Vol. 15, Issue 2 https://doi.org/10.1016/j.jmoldx.2012.09.003	journal	March 2013
Fast-forward genetics enabled by new sequencing technologies Schneeberger, Korbinian; Weigel, Detlef Trends in Plant Science, Vol. 16, Issue 5 https://doi.org/10.1016/j.tplants.2011.02.006	journal	May 2011
Advances and limits of using population genetics to understand local adaptation Tiffin, Peter; Ross-Ibarra, Jeffrey Trends in Ecology & Evolution, Vol. 29, Issue 12 https://doi.org/10.1016/j.tree.2014.10.004	journal	December 2014
Advances and Limits of Using Population Genetics to Understand Local Adaptation Tiffin, Peter; Ross-Ibarra, Jeffrey Trends in Ecology & Evolution, Vol. 32, Issue 10 https://doi.org/10.1016/j.tree.2017.08.001	journal	October 2017
Genome sequencing and analysis of the model grass Brachypodium distachyon No authors listed Nature, Vol. 463, Issue 7282, p. 763-768 https://doi.org/10.1038/nature08747	journal	February 2010
A map of human genome variation from population-scale sequencing No authors listed Nature, Vol. 467, Issue 7319, p. 1061-1073 https://doi.org/10.1038/nature09534	journal	October 2010
Sequencing technologies — the next generation Metzker, Michael L. Nature Reviews Genetics, Vol. 11, Issue 1 https://doi.org/10.1038/nrg2626	journal	December 2009
Ancient genomes reveal social and genetic structure of Late Neolithic Switzerland Furtwängler, Anja; Rohrlach, A. B.; Lamnidis, Thiseas C. Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-15560-x	journal	April 2020
Enhancements and modifications of primer design program Primer3 Koressaar, T.; Remm, M. Bioinformatics, Vol. 23, Issue 10 https://doi.org/10.1093/bioinformatics/btm091	journal	March 2007
Fast and accurate short read alignment with Burrows-Wheeler transform Li, H.; Durbin, R. Bioinformatics, Vol. 25, Issue 14 https://doi.org/10.1093/bioinformatics/btp324	journal	May 2009
The Sequence Alignment/Map format and SAMtools Li, H.; Handsaker, B.; Wysoker, A. Bioinformatics, Vol. 25, Issue 16 https://doi.org/10.1093/bioinformatics/btp352	journal	June 2009
Trimmomatic: a flexible trimmer for Illumina sequence data Bolger, Anthony M.; Lohse, Marc; Usadel, Bjoern Bioinformatics, Vol. 30, Issue 15 https://doi.org/10.1093/bioinformatics/btu170	journal	April 2014
Primer3—new capabilities and interfaces Untergasser, Andreas; Cutcutache, Ioana; Koressaar, Triinu Nucleic Acids Research, Vol. 40, Issue 15 https://doi.org/10.1093/nar/gks596	journal	June 2012
Application of Targeted Next-generation Sequencing, TruSeq Custom Amplicon Assay for Molecular Pathology Diagnostics on Formalin-fixed and Paraffin-embedded Samples Csernák, Erzsébet; Molnár, János; Tusnády, Gábor E. Applied Immunohistochemistry & Molecular Morphology, Vol. 25, Issue 7 https://doi.org/10.1097/PAI.0000000000000325	journal	January 2017
The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data McKenna, A.; Hanna, M.; Banks, E. Genome Research, Vol. 20, Issue 9 https://doi.org/10.1101/gr.107524.110	journal	July 2010
Discovery of Rare Mutations in Populations: TILLING by Sequencing Tsai, Helen; Howell, Tyson; Nitcher, Rebecca Plant Physiology, Vol. 156, Issue 3 https://doi.org/10.1104/pp.110.169748	journal	April 2011
Genotyping-in-Thousands by sequencing (GT-seq): A cost effective SNP genotyping method based on custom amplicon sequencing Campbell, Nathan R.; Harmon, Stephanie A.; Narum, Shawn R. Molecular Ecology Resources, Vol. 15, Issue 4 https://doi.org/10.1111/1755-0998.12357	journal	December 2014
Large-scale detection of rare variants via pooled multiplexed next-generation sequencing: towards next-generation Ecotilling: Detection of rare variants via next-generation sequencing Marroni, Fabio; Pinosio, Sara; Di Centa, Eleonora The Plant Journal, Vol. 67, Issue 4 https://doi.org/10.1111/j.1365-313X.2011.04627.x	journal	July 2011
Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing†: SNP discovery in rice starch genes by MPS technology Kharabian-Masouleh, Ardashir; Waters, Daniel L. E.; Reinke, Russell F. Plant Biotechnology Journal, Vol. 9, Issue 9 https://doi.org/10.1111/j.1467-7652.2011.00629.x	journal	June 2011
Ancient plant DNA: review and prospects: Research review Gugerli, Felix; Parducci, Laura; Petit, Rémy J. New Phytologist, Vol. 166, Issue 2 https://doi.org/10.1111/j.1469-8137.2005.01360.x	journal	February 2005
Molecular, morphological, and cytological analysis of diverse Brachypodium distachyon inbred lines Filiz, E.; Ozdemir, B. S.; Budak, F. Genome, Vol. 52, Issue 10 https://doi.org/10.1139/G09-062	journal	October 2009
User guide for mapping-by-sequencing in Arabidopsis James, Geo Velikkakam; Patel, Vipul; Nordström, Karl JV Genome Biology, Vol. 14, Issue 6 https://doi.org/10.1186/gb-2013-14-6-r61	journal	June 2013
Characterization and functional analysis of phytoene synthase gene family in tobacco Wang, Zhaojun; Zhang, Lin; Dong, Chen BMC Plant Biology, Vol. 21, Issue 1 https://doi.org/10.1186/s12870-020-02816-3	journal	January 2021
Stairway Plot 2: demographic history inference with folded SNP frequency spectra Liu, Xiaoming; Fu, Yun-Xin Genome Biology, Vol. 21, Issue 1 https://doi.org/10.1186/s13059-020-02196-9	journal	November 2020
Imputation of Unordered Markers and the Impact on Genomic Selection Accuracy Rutkoski, Jessica E.; Poland, Jesse; Jannink, Jean-Luc G3: Genes\|Genomes\|Genetics, Vol. 3, Issue 3 https://doi.org/10.1534/g3.112.005363	journal	March 2013
Genetic Diversity Analysis of Highly Incomplete SNP Genotype Data with Imputations: An Empirical Assessment Fu, Yong-Bi G3: Genes\|Genomes\|Genetics, Vol. 4, Issue 5 https://doi.org/10.1534/g3.114.010942	journal	March 2014
A map of human genome variation from population-scale sequencing Yun, Li, The University of North Carolina at Chapel Hill University Libraries https://doi.org/10.17615/med3-cd58	text	January 2010
The Quest for Rare Variants: Pooled Multiplexed Next Generation Sequencing in Plants Marroni, Fabio; Pinosio, Sara; Morgante, Michele Frontiers in Plant Science, Vol. 3 https://doi.org/10.3389/fpls.2012.00133	journal	January 2012
Next-Generation Sampling: Pairing Genomics with Herbarium Specimens Provides Species-Level Signal in Solidago (Asteraceae) Beck, James B.; Semple, John C. Applications in Plant Sciences, Vol. 3, Issue 6 https://doi.org/10.3732/apps.1500014	journal	June 2015
Genome sequencing and analysis of the model grass Brachypodium distachyon Initiative, International Brachypodium; Vogel, J. P.; Wicker, T. Nature Publishing Group https://doi.org/10.5167/uzh-43350	text	January 2010

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