Quantification of epigenetic biomarkers: an evaluation of established and emerging methods for DNA methylation analysis

BMC Genomics

Alison S Devonshire²  Carole A Foy²  Martin S Taylor¹  Jim F Huggett²  Nicholas Redshaw² 

[1] Medical and Developmental Genetics Section, MRC Human Genetics Unit, IGMM, University of Edinburgh, Edinburgh, UK;LGC, Queens Road, Teddington, Middlesex TW11 0LY, UK

关键词: Quantification;    NGS;    Digital PCR;    Reference material;    DNA methylation;   

Others  :  1121473
DOI  :  10.1186/1471-2164-15-1174

 received in 2014-11-12, accepted in 2014-12-10,  发布年份 2014

【 摘 要 】

Background

DNA methylation is an important epigenetic mechanism in several human diseases, most notably cancer. The quantitative analysis of DNA methylation patterns has the potential to serve as diagnostic and prognostic biomarkers, however, there is currently a lack of consensus regarding the optimal methodologies to quantify methylation status. To address this issue we compared five analytical methods: (i) MethyLight qPCR, (ii) MethyLight digital PCR (dPCR), methylation-sensitive and -dependent restriction enzyme (MSRE/MDRE) digestion followed by (iii) qPCR or (iv) dPCR, and (v) bisulfite amplicon next generation sequencing (NGS). The techniques were evaluated for linearity, accuracy and precision.

Results

MethyLight qPCR displayed the best linearity across the range of tested samples. Observed methylation measured by MethyLight- and MSRE/MDRE-qPCR and -dPCR were not significantly different to expected values whilst bisulfite amplicon NGS analysis over-estimated methylation content. Bisulfite amplicon NGS showed good precision, whilst the lower precision of qPCR and dPCR analysis precluded discrimination of differences of < 25% in methylation status. A novel dPCR MethyLight assay is also described as a potential method for absolute quantification that simultaneously measures both sense and antisense DNA strands following bisulfite treatment.

Conclusions

Our findings comprise a comprehensive benchmark for the quantitative accuracy of key methods for methylation analysis and demonstrate their applicability to the quantification of circulating tumour DNA biomarkers by using sample concentrations that are representative of typical clinical isolates.

【 授权许可】

   
2014 Redshaw et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Robertson KD: DNA methylation and human disease. Nat Rev Genet 2005, 6(8):597-610.
• [2]Jones PA, Baylin SB: The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002, 3(6):415-428.
• [3]Irizarry RA, Ladd-Acosta C, Wen B, Wu Z, Montano C, Onyango P, Cui H, Gabo K, Rongione M, Webster M, Ji H, Potash JB, Sabunciyan S, Feinberg AP: The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores. Nat Genet 2009, 41(2):178-186.
• [4]Rakyan VK, Down TA, Balding DJ, Beck S: Epigenome-wide association studies for common human diseases. Nat Rev Genet 2011, 12(8):529-541.
• [5]Issa JP: CpG island methylator phenotype in cancer. Nat Rev Cancer 2004, 4(12):988-993.
• [6]Brooks J, Cairns P, Zeleniuch-Jacquotte A: Promoter methylation and the detection of breast cancer. Cancer Causes Control 2009, 20(9):1539-1550.
• [7]Fleischhacker M, Schmidt B: Circulating nucleic acids (CNAs) and cancer–a survey. Biochim Biophys Acta 2007, 1775(1):181-232.
• [8]Laird PW: Principles and challenges of genomewide DNA methylation analysis. Nat Rev Genet 2010, 11(3):191-203.
• [9]Kristensen LS, Hansen LL: PCR-based methods for detecting single-locus DNA methylation biomarkers in cancer diagnostics, prognostics, and response to treatment. Clin Chem 2009, 55(8):1471-1483.
• [10]Hayatsu H, Wataya Y, Kai K, Iida S: Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry 1970, 9(14):2858-2865.
• [11]Hayatsu H, Wataya Y, Kazushige K: The addition of sodium bisulfite to uracil and to cytosine. J Am Chem Soc 1970, 92(3):724-726.
• [12]Shiraishi M, Hayatsu H: High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation. DNA Res 2004, 11(6):409-415.
• [13]Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL: A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 1992, 89(5):1827-1831.
• [14]Korshunova Y, Maloney RK, Lakey N, Citek RW, Bacher B, Budiman A, Ordway JM, McCombie WR, Leon J, Jeddeloh JA, McPherson JD: Massively parallel bisulphite pyrosequencing reveals the molecular complexity of breast cancer-associated cytosine-methylation patterns obtained from tissue and serum DNA. Genome Res 2008, 18(1):19-29.
• [15]Potapova A, Albat C, Hasemeier B, Haeussler K, Lamprecht S, Suerbaum S, Kreipe H, Lehmann U: Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution. BMC Biotechnol 2011, 11:6. BioMed Central Full Text
• [16]Taylor KH, Kramer RS, Davis JW, Guo J, Duff DJ, Xu D, Caldwell CW, Shi H: Ultradeep bisulfite sequencing analysis of DNA methylation patterns in multiple gene promoters by 454 sequencing. Cancer Res 2007, 67(18):8511-8518.
• [17]Tost J, Gut IG: DNA methylation analysis by pyrosequencing. Nat Protoc 2007, 2(9):2265-2275.
• [18]Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, Danenberg PV, Laird PW: MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 2000, 28(8):E32.
• [19]Trinh BN, Long TI, Laird PW: DNA methylation analysis by MethyLight technology. Methods 2001, 25(4):456-462.
• [20]Weisenberger DJ, Trinh BN, Campan M, Sharma S, Long TI, Ananthnarayan S, Liang G, Esteva FJ, Hortobagyi GN, McCormick F, Jones PA, Laird PW: DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight. Nucleic Acids Res 2008, 36(14):4689-4698.
• [21]Vogelstein B, Kinzler KW: Digital PCR. Proc Natl Acad Sci U S A 1999, 96(16):9236-9241.
• [22]Corbisier P, Bhat S, Partis L, Xie VR, Emslie KR: Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction. Anal Bioanal Chem 2010, 396(6):2143-2150.
• [23]Oakes CC, La Salle S, Robaire B, Trasler JM: Evaluation of a quantitative DNA methylation analysis technique using methylation-sensitive/dependent restriction enzymes and real-time PCR. Epigenetics 2006, 1(3):146-152.
• [24]Hashimoto K, Kokubun S, Itoi E, Roach HI: Improved quantification of DNA methylation using methylation-sensitive restriction enzymes and real-time PCR. Epigenetics 2007, 2(2):86-91.
• [25]Holemon H, Korshunova Y, Ordway JM, Bedell JA, Citek RW, Lakey N, Leon J, Finney M, McPherson JD, Jeddeloh JA: MethylScreen: DNA methylation density monitoring using quantitative PCR. Biotechniques 2007, 43(5):683-693.
• [26]Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP, et al.: High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 2011, 83(22):8604-8610.
• [27]Tong YK, Jin S, Chiu RW, Ding C, Chan KC, Leung TY, Yu L, Lau TK, Lo YM: Noninvasive prenatal detection of trisomy 21 by an epigenetic-genetic chromosome-dosage approach. Clin Chem 2010, 56(1):90-98.
• [28]Robertson KD, Jones PA: The human ARF cell cycle regulatory gene promoter is a CpG island which can be silenced by DNA methylation and down-regulated by wild-type p53. Mol Cell Biol 1998, 18(11):6457-6473.
• [29]Zheng S, Chen P, McMillan A, Lafuente A, Lafuente MJ, Ballesta A, Trias M, Wiencke JK: Correlations of partial and extensive methylation at the p14(ARF) locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors. Carcinogenesis 2000, 21(11):2057-2064.
• [30]Shen L, Kondo Y, Hamilton SR, Rashid A, Issa JP: P14 methylation in human colon cancer is associated with microsatellite instability and wild-type p53. Gastroenterology 2003, 124(3):626-633.
• [31]Furonaka O, Takeshima Y, Awaya H, Ishida H, Kohno N, Inai K: Aberrant methylation of p14(ARF), p15(INK4b) and p16(INK4a) genes and location of the primary site in pulmonary squamous cell carcinoma. Pathol Int 2004, 54(8):549-555.
• [32]Kawamoto K, Enokida H, Gotanda T, Kubo H, Nishiyama K, Kawahara M, Nakagawa M: p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer. Biochem Biophys Res Commun 2006, 339(3):790-796.
• [33]Sato F, Harpaz N, Shibata D, Xu Y, Yin J, Mori Y, Zou TT, Wang S, Desai K, Leytin A, Selaru FM, Abraham JM, Meltzer SJ: Hypermethylation of the p14(ARF) gene in ulcerative colitis-associated colorectal carcinogenesis. Cancer Res 2002, 62(4):1148-1151.
• [34]Lee M, Sup Han W, Kyoung Kim O, Hee Sung S, Sun Cho M, Lee SN, Koo H: Prognostic value of p16INK4a and p14ARF gene hypermethylation in human colon cancer. Pathol Res Pract 2006, 202(6):415-424.
• [35]Ishida E, Nakamura M, Ikuta M, Shimada K, Matsuyoshi S, Kirita T, Konishi N: Promotor hypermethylation of p14ARF is a key alteration for progression of oral squamous cell carcinoma. Oral Oncol 2005, 41(6):614-622.
• [36]Hsu HS, Wang YC, Tseng RC, Chang JW, Chen JT, Shih CM, Chen CY: 5' cytosine-phospho-guanine island methylation is responsible for p14ARF inactivation and inversely correlates with p53 overexpression in resected non-small cell lung cancer. Clin Cancer Res 2004, 10(14):4734-4741.
• [37]Devonshire AS, Whale AS, Gutteridge A, Jones G, Cowen S, Foy CA, Huggett JF: Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Anal Bioanal Chem 2014, 406(26):6499-6512.
• [38]Bhat S, Curach N, Mostyn T, Bains GS, Griffiths KR, Emslie KR: Comparison of methods for accurate quantification of DNA mass concentration with traceability to the international system of units. Anal Chem 2010, 82(17):7185-7192.
• [39]Grunau C, Clark SJ, Rosenthal A: Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 2001, 29(13):E65-65.
• [40]Ogino S, Kawasaki T, Brahmandam M, Cantor M, Kirkner GJ, Spiegelman D, Makrigiorgos GM, Weisenberger DJ, Laird PW, Loda M, et al.: Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J Mol Diagn 2006, 8(2):209-217.
• [41]Weaver S, Dube S, Mir A, Qin J, Sun G, Ramakrishnan R, Jones RC, Livak KJ: Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution. Methods 2010, 50(4):271-276.
• [42]Hayden RT, Gu Z, Ingersoll J, Abdul-Ali D, Shi L, Pounds S, Caliendo AM: Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus. J Clin Microbiol 2013, 51(2):540-546.
• [43]Quillien V, Lavenu A, Karayan-Tapon L, Carpentier C, Labussiere M, Lesimple T, Chinot O, Wager M, Honnorat J, Saikali S, Fina F, Sanson M, Figarella-Branger D: Comparative assessment of 5 methods (methylation-specific polymerase chain reaction, MethyLight, pyrosequencing, methylation-sensitive high-resolution melting, and immunohistochemistry) to analyze O6-methylguanine-DNA-methyltranferase in a series of 100 glioblastoma patients. Cancer 2012, 118(17):4201-4211.
• [44]Huse S, Huber J, Morrison H, Sogin M, Welch D: Accuracy and quality of massively parallel DNA pyrosequencing. Genome Biology 2007, 8(7):R143. BioMed Central Full Text
• [45]Warnecke PM, Stirzaker C, Melki JR, Millar DS, Paul CL, Clark SJ: Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res 1997, 25(21):4422-4426.
• [46]Wojdacz TK, Hansen LL: Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay. Biotechniques 2006, 41(3):274. 276, 278
• [47]Wojdacz TK, Hansen LL, Dobrovic A: A new approach to primer design for the control of PCR bias in methylation studies. BMC Res Notes 2008, 1:54. BioMed Central Full Text
• [48]Dabney J, Meyer M: Length and GC-biases during sequencing library amplification: a comparison of various polymerase-buffer systems with ancient and modern DNA sequencing libraries. Biotechniques 2012, 52(2):87-94.
• [49]Shen L, Guo Y, Chen X, Ahmed S, Issa JP: Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. Biotechniques 2007, 42(1):48. 50, 52 passim
• [50]Berry D, Ben Mahfoudh K, Wagner M, Loy A: Barcoded primers used in multiplex amplicon pyrosequencing bias amplification. Appl Environ Microbiol 2011, 77(21):7846-7849.
• [51]Gries J, Schumacher D, Arand J, Lutsik P, Markelova MR, Fichtner I, Walter J, Sers C, Tierling S: Bi-PROF: bisulfite profiling of target regions using 454 GS FLX Titanium technology. Epigenetics 2013, 8(7):765-771.
• [52]Oakes CC, La Salle S, Trasler JM, Robaire B: Restriction digestion and real-time PCR (qAMP). Methods Mol Biol 2009, 507:271-280.
• [53]Dube S, Qin J, Ramakrishnan R: Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device. PLoS One 2008, 3(8):e2876.
• [54]Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T, Fuchs CS, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT, Bustin SA: The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments. Clin Chem 2013, 59(6):892-902.
• [55]Whale AS, Huggett JF, Cowen S, Speirs V, Shaw J, Ellison S, Foy CA, Scott DJ: Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res 2012, 40(11):e82.
• [56]Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, Fiala E, Ehrlich M, Laird PW: Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res 2005, 33(21):6823-6836.
• [57]Widschwendter M, Siegmund KD, Muller HM, Fiegl H, Marth C, Muller-Holzner E, Jones PA, Laird PW: Association of breast cancer DNA methylation profiles with hormone receptor status and response to tamoxifen. Cancer Res 2004, 64(11):3807-3813.
• [58]Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT: Primer sequence disclosure: a clarification of the MIQE guidelines. Clin Chem 2011, 57(6):919-921.