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.
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References
Ahn SJ, Costa J, Emanuel JR (1996) PicoGreen quantitation of DNA: effective evaluation of samples pre- or post-PCR. Nucleic Acids Res 24:2623–2625
Albert TJ, Molla MN, Muzny DM, Nazareth L, Wheeler D, Song XZ, Richmond TA, Middle CM, Rodesch MJ, Packard CJ, Weinstock GM, Gibbs RA (2007) Direct selection of human genomic loci by microarray hybridization. Nat Methods 4:903–905
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Ashlock D, Guo L, Qiu F (2002) Greedy closure evolutionary algorithms. In: Proceedings of the 2002 congress on evolutionary computation, pp 1296–1301
Bainbridge MN, Wang M, Burgess DL, Kovar C, Rodesch MJ, D’Ascenzo M, Kitzman J, Wu YQ, Newsham I, Richmond TA, Jeddeloh JA, Muzny D, Albert TJ, Gibbs RA (2010) Whole exome capture in solution with 3 Gbp of data. Genome Biol 11:R62
Bashiardes S, Veile R, Helms C, Mardis ER, Bowcock AM, Lovett M (2005) Direct genomic selection. Nat Methods 2:63–69
Bentley DR (2006) Whole-genome re-sequencing. Curr Opin Genet Dev 16:545–552
Binladen J, Gilbert MTP, Bollback JP, Panitz F, Bendixen C, Nielsen R, Willerslev E (2007) The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing. PLoS ONE 2:e197. doi:10.1371/journal.pone.0000197
Bodi K, Perera AG, Adams PS, Bintzler D, Dewar K, Grove DS, Kieleczawa J, Lyons RH, Neubert TA, Noll AC, Singh S, Steen R, Zianni M (2013) Comparison of commercially available target enrichment methods for next-generation sequencing. J Biomol Tech 24:73–86
Choi M, Scholl UI, Ji W, Liu T, Tikhonova IR, Zumbo P, Nayir A, Bakkaloglu A, Ozen S, Sanjad S, Nelson-Williams C, Farhi A, Mane S, Lifton RP (2009) Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci USA 106:19096–19101
Craig DW, Pearson JV, Szelinger S, Sekar A, Redman M, Corneveaux JJ, Pawlowski TL, Laub T, Nunn G, Stephan DA, Homer N, Huentelman MJ (2008) Identification of genetic variants using bar-coded multiplexed sequencing. Nat Methods 5:887–893
Dahl F, Stenberg J, Fredriksson S, Welch K, Zhang M, Nilsson M, Bicknell D, Bodmer WF, Davis RW, Ji HL (2007) Multigene amplification and massively parallel sequencing for cancer mutation discovery. Proc Natl Acad Sci USA 104:9387–9392
Fu Y, Springer NM, Gerhardt DJ, Ying K, Yeh CT, Wu W, Swanson-Wagner R, D’Ascenzo M, Millard T, Freeberg L, Aoyama N, Kitzman J, Burgess D, Richmond T, Albert TJ, Barbazuk WB, Jeddeloh JA, Schnable PS (2010) Repeat subtraction-mediated sequence capture from a complex genome. Plant J Cell Mol Biol 62:898–909
Gnirke A, Melnikov A, Maguire J, Rogov P, LeProust EM, Brockman W, Fennell T, Giannoukos G, Fisher S, Russ C, Gabriel S, Jaffe DB, Lander ES, Nusbaum C (2009) Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol 27:182–189
Gribbon BM, Pearce SR, Kalendar R, Schulman AH, Paulin L, Jack P, Kumar A, Flavell AJ (1999) Phylogeny and transpositional activity of Ty1-copia group retrotransposons in cereal genomes. Mol Gen Genetics 261:883–891
Hopp K, Heyer CM, Hommerding CJ, Henke SA, Sundsbak JL, Patel S, Patel P, Consugar MB, Czarnecki PG, Gliem TJ, Torres VE, Rossetti S, Harris PC (2011) B9D1 is revealed as a novel Meckel syndrome (MKS) gene by targeted exon-enriched next-generation sequencing and deletion analysis. Hum Mol Genet 20:2524–2534
Huang X, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–877
Klock HE, Lesley SA (2009) The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis. Methods Mol Biol 498:91–103
Lee H, O’Connor BD, Merriman B, Funari VA, Homer N, Chen Z, Cohn DH, Nelson SF (2009) Improving the efficiency of genomic loci capture using oligonucleotide arrays for high throughput resequencing. BMC Genom 10:646
Mamanova L, Coffey AJ, Scott CE, Kozarewa I, Turner EH, Kumar A, Howard E, Shendure J, Turner DJ (2010) Target-enrichment strategies for next-generation sequencing. Nat Methods 7:111–118
Mardis ER (2008) Next-generation DNA sequencing methods. Annu Rev Genom Hum G 9:387–402
Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer ML, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu P, Begley RF, Rothberg JM (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376–380
Milne I, Stephen G, Bayer M, Cock PJA, Pritchard L, Cardle L, Shaw PD, Marshall D (2013) Using Tablet for visual exploration of second-generation sequencing data. Brief Bioinform 14:193–202
Okou DT, Steinberg KM, Middle C, Cutler DJ, Albert TJ, Zwick ME (2007) Microarray-based genomic selection for high-throughput resequencing. Nat Methods 4:907–909
Porreca GJ, Zhang K, Li JB, Xie B, Austin D, Vassallo SL, LeProust EM, Peck BJ, Emig CJ, Dahl F, Gao Y, Church GM, Shendure J (2007) Multiplex amplification of large sets of human exons. Nat Methods 4:931–936
Qiu F, Guo L, Wen TJ, Liu F, Ashlock DA, Schnable PS (2003) DNA sequence-based “bar codes” for tracking the origins of expressed sequence tags from a maize cDNA library constructed using multiple mRNA sources. Plant Physiol 133:475–481
Rostoks N, Mudie S, Cardle L, Russell J, Ramsay L, Booth A, Svensson JT, Wanamaker SI, Walia H, Rodriguez EM, Hedley PE, Liu H, Morris J, Close TJ, Marshall DF, Waugh R (2005) Genome-wide SNP discovery and linkage analysis in barley based on genes responsive to abiotic stress. Mol Genetics Genomics 274:515–527
SanMiguel P, Tikhonov A, Jin YK, Motchoulskaia N, Zakharov D, Melake-Berhan A, Springer PS, Edwards KJ, Lee M, Avramova Z, Bennetzen JL (1996) Nested retrotransposons in the intergenic regions of the maize genome. Science 274:765–768
Smith AM, Heisler LE, St Onge RP, Farias-Hesson E, Wallace IM, Bodeau J, Harris AN, Perry KM, Giaever G, Pourmand N, Nislow C (2010) Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples. Nucleic Acids Res 38:e142. doi:10.1093/nar/gkq368
Tan IB, Cutcutache I, Zang ZJ, Iqbal J, Yap SF, Hwang W, Lim WT, Teh BT, Rozen S, Tan EH, Tan P (2011) Fanconi’s anemia in adulthood: chemoradiation-induced bone marrow failure and a novel FANCA mutation identified by targeted deep sequencing. J Clin Oncol Off J Am Soc Clin Oncol 29:e591–e594
Tewhey R, Warner JB, Nakano M, Libby B, Medkova M, David PH, Kotsopoulos SK, Samuels ML, Hutchison JB, Larson JW, Topol EJ, Weiner MP, Harismendy O, Olson J, Link DR, Frazer KA (2009) Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol 27:U1025–U1094
Turner EH, Lee CL, Ng SB, Nickerson DA, Shendure J (2009) Massively parallel exon capture and library-free resequencing across 16 genomes. Nat Methods 6:315–316
Waugh R, McLean K, Flavell AJ, Pearce SR, Kumar A, Thomas BB, Powell W (1997) Genetic distribution of Bare-1-like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol Gen Genetics 253:687–694
Acknowledgments
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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Ho, T., Cardle, L., Xu, X. et al. Genome-Tagged Amplification (GTA): a PCR-based method to prepare sample-tagged amplicons from hundreds of individuals for next generation sequencing. Mol Breeding 34, 977–988 (2014). https://doi.org/10.1007/s11032-014-0090-7
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DOI: https://doi.org/10.1007/s11032-014-0090-7