High-throughput single nucleotide polymorphism genotyping using nanofluidic Dynamic Arrays
Tóm tắt
Single nucleotide polymorphisms (SNPs) have emerged as the genetic marker of choice for mapping disease loci and candidate gene association studies, because of their high density and relatively even distribution in the human genomes. There is a need for systems allowing medium multiplexing (ten to hundreds of SNPs) with high throughput, which can efficiently and cost-effectively generate genotypes for a very large sample set (thousands of individuals). Methods that are flexible, fast, accurate and cost-effective are urgently needed. This is also important for those who work on high throughput genotyping in non-model systems where off-the-shelf assays are not available and a flexible platform is needed. We demonstrate the use of a nanofluidic Integrated Fluidic Circuit (IFC) - based genotyping system for medium-throughput multiplexing known as the Dynamic Array, by genotyping 994 individual human DNA samples on 47 different SNP assays, using nanoliter volumes of reagents. Call rates of greater than 99.5% and call accuracies of greater than 99.8% were achieved from our study, which demonstrates that this is a formidable genotyping platform. The experimental set up is very simple, with a time-to-result for each sample of about 3 hours. Our results demonstrate that the Dynamic Array is an excellent genotyping system for medium-throughput multiplexing (30-300 SNPs), which is simple to use and combines rapid throughput with excellent call rates, high concordance and low cost. The exceptional call rates and call accuracy obtained may be of particular interest to those working on validation and replication of genome- wide- association (GWA) studies.
Tài liệu tham khảo
Carlson CS, Eberle MA, Kruglyak L, Nickerson DA: Mapping complex disease loci in whole-genome association studies. Nature. 2004, 429 (6990): 446-452.
Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, et al: Initial sequencing and analysis of the human genome. Nature. 2001, 409 (6822): 860-921.
Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, et al: The sequence of the human genome. Science. 2001, 291 (5507): 1304-1351.
Consortium IHGS: Finishing the euchromatic sequence of the human genome. Nature. 2004, 431 (7011): 931-945.
Consortium TIH: The International HapMap Project. Nature. 2003, 426 (6968): 789-796.
Consortium IH: A haplotype map of the human genome. Nature. 2005, 437 (7063): 1299-1320.
Frazer KA, Ballinger DG, Cox DR, Hinds DA, Stuve LL, Gibbs RA, Belmont JW, Boudreau A, Hardenbol P, Leal SM, et al: A second generation human haplotype map of over 3.1 million SNPs. Nature. 2007, 449 (7164): 851-861.
Hirschhorn JN, Daly MJ: Genome-wide association studies for common diseases and complex traits. Nat Rev Genet. 2005, 6 (2): 95-108.
Duerr RH, Taylor KD, Brant SR, Rioux JD, Silverberg MS, Daly MJ, Steinhart AH, Abraham C, Regueiro M, Griffiths A, et al: A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science. 2006, 314 (5804): 1461-1463.
Frayling TM, Timpson NJ, Weedon MN, Zeggini E, Freathy RM, Lindgren CM, Perry JR, Elliott KS, Lango H, Rayner NW, et al: A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science. 2007, 316 (5826): 889-894.
Hunter DJ, Kraft P, Jacobs KB, Cox DG, Yeager M, Hankinson SE, Wacholder S, Wang Z, Welch R, Hutchinson A, et al: A genome-wide association study identifies alleles in FGFR2 associated with risk of sporadic postmenopausal breast cancer. Nat Genet. 2007, 39 (7): 870-874.
Thomas G, Jacobs KB, Kraft P, Yeager M, Wacholder S, Cox DG, Hankinson SE, Hutchinson A, Wang Z, Yu K, et al: A multistage genome-wide association study in breast cancer identifies two new risk alleles at 1p11.2 and 14q24.1(RAD51L1). Nat Genet. 2009, 41 (5): 579-584.
Thomas G, Jacobs KB, Yeager M, Kraft P, Wacholder S, Orr N, Yu K, Chatterjee N, Welch R, Hutchinson A, et al: Multiple loci identified in a genome-wide association study of prostate cancer. Nat Genet. 2008, 40 (3): 310-315.
McCarthy MI, Abecasis GR, Cardon LR, Goldstein DB, Little J, Ioannidis JP, Hirschhorn JN: Genome-wide association studies for complex traits: consensus, uncertainty and challenges. Nat Rev Genet. 2008, 9 (5): 356-369.
Kwok PY: Methods for genotyping single nucleotide polymorphisms. Annu Rev Genomics Hum Genet. 2001, 2: 235-258.
Chen X, Sullivan PF: Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput. Pharmacogenomics J. 2003, 3 (2): 77-96.
Matsuzaki H, Dong S, Loi H, Di X, Liu G, Hubbell E, Law J, Berntsen T, Chadha M, Hui H, et al: Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nat Methods. 2004, 1 (2): 109-111.
Hardenbol P, Yu F, Belmont J, Mackenzie J, Bruckner C, Brundage T, Boudreau A, Chow S, Eberle J, Erbilgin A, et al: Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay. Genome Res. 2005, 15 (2): 269-275.
Murray SS, Oliphant A, Shen R, McBride C, Steeke RJ, Shannon SG, Rubano T, Kermani BG, Fan JB, Chee MS, et al: A highly informative SNP linkage panel for human genetic studies. Nat Methods. 2004, 1 (2): 113-117.
Livak KJ, Marmaro J, Todd JA: Towards fully automated genome-wide polymorphism screening. Nat Genet. 1995, 9 (4): 341-342.
Tyagi S, Bratu DP, Kramer FR: Multicolor molecular beacons for allele discrimination. Nat Biotechnol. 1998, 16 (1): 49-53.
Spurgeon SL, Jones RC, Ramakrishnan R: High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PLoS ONE. 2008, 3 (2): e1662-
Andriole GL, Reding D, Hayes RB, Prorok PC, Gohagan JK: The prostate, lung, colon, and ovarian (PLCO) cancer screening trial: Status and promise. Urol Oncol. 2004, 22 (4): 358-361.
Qin J, Jones RC, Ramakrishnan R: Studying copy number variations using a nanofluidic platform. Nucleic Acids Res. 2008, 36 (18): e116-
Product Literature for GoldenGate® Assay. [http://www.illumina.com/Documents/products/workflows/workflow_goldengate_assay.pdf]
Ehrich M, Böcker S, Boom van den D: Multiplexed discovery of sequence polymorphisms using base-specific cleavage and MALDI-TOF MS. Nucleic Acids Res. 2005, 33 (4): e38-
Seeb JE, Pascal CE, Ramakrishnan R, Seeb LW: SNP Genotyping by the 5'-nuclease Reaction: Advances in High Throughput Genotyping with Non-Model Organisms. Methods Mol Biol. 578: 277-92.