Abstract
Owing to the high throughput and low cost, next generation sequencing has attracted much attention for SNP genotyping application for researchers. Here, we introduce a new method based on three-round multiplex PCR to precisely genotype SNPs with next generation sequencing. This method can as much as possible consume the equivalent amount of each pair of specific primers to largely eliminate the amplification discrepancy between different loci. After the PCR amplification, the products can be directly subjected to next generation sequencing platform. We simultaneously amplified 37 SNP loci of 757 samples and sequenced all amplicons on ion torrent PGM platform; 90.5 % of the target SNP loci were accurately genotyped (at least 15×) and 90.4 % amplicons had uniform coverage with a variation less than 50-fold. Ligase detection reaction (LDR) was performed to genotype the 19 SNP loci (as part of the 37 SNP loci) with 91 samples randomly selected from the 757 samples, and 99.5 % genotyping data were consistent with the next generation sequencing results. Our results demonstrate that three-round PCR coupled with next generation sequencing is an efficient and economical genotyping approach.
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Shavrukov Y, Suchecki R, Eliby S, Abugalieva A, Kenebayev S, Langridge P. Application of next-generation sequencing technology to study genetic diversity and identify unique SNP markers in bread wheat from Kazakhstan. BMC Plant Biol. 2014;14:258.
Li R, Li Y, Fang X, Yang H, Wang J, Kristiansen K, Wang J. SNP detection for massively parallel whole-genome resequencing. Genome Res. 2009;19(6):1124–32.
Syvanen AC. Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev Genet. 2001;2(12):930–42.
Li R, Fan W, Tian G, Zhu H, He L, Cai J, Huang Q, Cai Q, Li B, Bai Y, Zhang Z, Zhang Y, Wang W, Li J, Wei F, Li H, Jian M, Li J, Zang Z, Nielsen R, Li D, Gu W, Yang Z, Xuan Z, Ryder OA, Leung FC, Zhou Y, Cao J, Sun X, Fu Y, Fang X, Guo X, Wang B, Hou R, Shen F, Mu B, Ni P, Lin R, Qian W, Wang G, Yu C, Nie W, Wang J, Wu Z, Liang H, Min J, Wu Q, Cheng S, Ruan J, Wang M, Shi Z, Wen M, Liu B, Ren X, Zheng H, Dong D, Cook K, Shan G, Zhang H, Kosiol C, Xie X, Lu Z, Zheng H, Li Y, Steiner CC, Lam TT, Lin S, Zhang Q, Li G, Tian J, Gong T, Liu H, Zhang D, Fang L, Ye C, Zhang J, Hu W, Xu A, Ren Y, Zhang G, Bruford MW, Li Q, Ma L, Guo Y, An N, Hu Y, Zheng Y, Shi Y, Li Z, Liu Q, Chen Y, Zhao J, Qu N, Zhao S, Tian F, Wang X, Wang H, Xu L, Liu X, Vinar T, Wang Y, Lam TW, Yiu SM, Liu S, Zhang H, Li D, Huang Y, Wang X, Yang G, Jiang Z, Wang J, Qin N, Li L, Li J, Bolund L, Kristiansen K, Wong GK, Olson M, Zhang X, Li S, Yang H, Wang J, Wang J. The sequence and de novo assembly of the giant panda genome. Nature. 2010;463(7279):311–7.
Xu X, Liu X, Ge S, Jensen JD, Hu F, Li X, Dong Y, Gutenkunst RN, Fang L, Huang L, Li J, He W, Zhang G, Zheng X, Zhang F, Li Y, Yu C, Kristiansen K, Zhang X, Wang J, Wright M, McCouch S, Nielsen R, Wang J, Wang W. Resequencing 50 accessions of cultivated and wild rice yields markers for identifying agronomically important genes. Nat Biotechnol. 2012;30(1):105–11.
Davey JW, Hohenlohe PA, Etter PD, Boone JQ, Catchen JM, Blaxter ML. Genome-wide genetic marker discovery and genotyping using next-generation sequencing. Nat Rev Genet. 2011;12(7):499–510.
Liu L, Li Y, Li S, Hu N, He Y, Pong R, Lin D, Lu L, Law M. Comparison of next-generation sequencing systems. J Biomed Biotechnol. 2012;2012:251364.
Campbell NR, Harmon SA, Narum SR. Genotyping-in-Thousands by sequencing (GT-seq): a cost effective SNP genotyping method based on custom amplicon sequencing. Mol Ecol Resour. 2015;15(4):855–67.
Boland JF, Chung CC, Roberson D, Mitchell J, Zhang X, Im KM, He J, Chanock SJ, Yeager M, Dean M. The new sequencer on the block: comparison of Life Technology's Proton sequencer to an Illumina HiSeq for whole-exome sequencing. Hum Genet. 2013;132(10):1153–63.
Genomes Project C, Abecasis GR, Altshuler D, Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA. A map of human genome variation from population-scale sequencing. Nature. 2010;467(7319):1061–73.
Sjoblom T, Jones S, Wood LD, Parsons DW, Lin J, Barber TD, Mandelker D, Leary RJ, Ptak J, Silliman N, Szabo S, Buckhaults P, Farrell C, Meeh P, Markowitz SD, Willis J, Dawson D, Willson JK, Gazdar AF, Hartigan J, Wu L, Liu C, Parmigiani G, Park BH, Bachman KE, Papadopoulos N, Vogelstein B, Kinzler KW, Velculescu VE. The consensus coding sequences of human breast and colorectal cancers. Science. 2006;314(5797):268–74.
Craig DW, Pearson JV, Szelinger S, Sekar A, Redman M, Corneveaux JJ, Pawlowski TL, Laub T, Nunn G, Stephan DA, Homer N, Huentelman MJ. Identification of genetic variants using bar-coded multiplexed sequencing. Nat Methods. 2008;5(10):887–93.
Meuzelaar LS, Lancaster O, Pasche JP, Kopal G, Brookes AJ. MegaPlex PCR: a strategy for multiplex amplification. Nat Methods. 2007;4(10):835–7.
Gnirke A, Melnikov A, Maguire J, Rogov P, LeProust EM, Brockman W, et al. Solution hybrid selection with ultra-long oligonucleotides for massively parallel targeted sequencing. Nat Biotechnol. 2009;27(2):182–9.
Albert TJ, Molla MN, Muzny DM, Nazareth L, Wheeler D, Song X, Richmond TA, Middle CM, Rodesch MJ, Packard CJ, Weinstock GM, Gibbs RA. Direct selection of human genomic loci by microarray hybridization. Nat Methods. 2007;4(11):903–5.
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. Multiplex amplification of large sets of human exons. Nat Methods. 2007;4(11):931–6.
Varley KE, Mitra RD. Nested Patch PCR enables highly multiplexed mutation discovery in candidate genes. Genome Res. 2008;18(11):1844–50.
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. Microdroplet-based PCR enrichment for large-scale targeted sequencing. Nat Biotechnol. 2009;27(11):1025–31.
Smith DR, Quinlan AR, Peckham HE, Makowsky K, Tao W, Woolf B, Shen L, Donahue WF, Tusneem N, Stromberg MP, Stewart DA, Zhang L, Ranade SS, Warner JB, Lee CC, Coleman BE, Zhang Z, McLaughlin SF, Malek JA, Sorenson JM, Blanchard AP, Chapman J, Hillman D, Chen F, Rokhsar DS, McKernan KJ, Jeffries TW, Marth GT, Richardson PM. Rapid whole-genome mutational profiling using next-generation sequencing technologies. Genome Res. 2008;18(10):1638–42.
Nielsen R, Paul JS, Albrechtsen A, Song YS. Genotype and SNP calling from next-generation sequencing data. Nat Rev Genet. 2011;12(6):443–51.
Sims D, Sudbery I, Ilott NE, Heger A, Ponting CP. Sequencing depth and coverage: key considerations in genomic analyses. Nat Rev Genet. 2014;15(2):121–32.
Hedges DJ, Burges D, Powell E, Almonte C, Huang J, Young S, Boese B, Schmidt M, Pericak-Vance MA, Martin E, Zhang X, Harkins TT, Zuchner S. Exome sequencing of a multigenerational human pedigree. PLoS One. 2009;4(12):e8232.
Sie D, Snijders PJ, Meijer GA, Doeleman MW, van Moorsel MI, van Essen HF, Eijk PP, Grunberg K, van Grieken NC, Thunnissen E, Verheul HM, Smit EF, Ylstra B, Heideman DA. Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology. Cell Oncol. 2014;37(5):353–61.
Tsongalis GJ, Peterson JD, de Abreu FB, Tunkey CD, Gallagher TL, Strausbaugh LD, Wells WA, Amos C I. Routine use of the Ion Torrent AmpliSeq Cancer Hotspot Panel for identification of clinically actionable somatic mutations. Clin Chem Lab Med. 2014;52(5):707–14.
Nguyen-Dumont T, Pope BJ, Hammet F, Southey MC, Park DJ. A high-plex PCR approach for massively parallel sequencing. BioTech. 2013;55(2):69–74.
Teer JK, Bonnycastle LL, Chines PS, Hansen NF, Aoyama N, Swift AJ, Abaan HO, Albert TJ. Nisc Comparative Sequencing Program. Margulies EH. Green ED. Collins FS. Mullikin JC. Biesecker LG. Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing. Genome Res. 2010;20(10):1420–31.
Turner EH, Lee C, Ng SB, Nickerson DA, Shendure J. Massively parallel exon capture and library-free resequencing across 16 genomes. Nat Methods. 2009;6(5):315–6.
Acknowledgments
The authors acknowledge support for this work by grants from the National Natural Science Foundation of China (no.31371257) and the Key Project of Science and Technology Commission of Shanghai Municipality (no.14140900502).
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Chen, K., Zhou, Yx., Li, K. et al. A novel three-round multiplex PCR for SNP genotyping with next generation sequencing. Anal Bioanal Chem 408, 4371–4377 (2016). https://doi.org/10.1007/s00216-016-9536-6
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DOI: https://doi.org/10.1007/s00216-016-9536-6