Genome-wide measures of DNA methylation in peripheral blood and the risk of urothelial cell carcinoma: A prospective nested case-control study

British Journal of Cancer

Volumn 115, Issue 6, 2016, Pages 664-673

  Dugué, Pierre Antoine ^a,b   Brinkman, Maree T ^a   Milne, Roger L ^a,b   Wong, Ee Ming ^c   FitzGerald, Liesel M ^a,d   Bassett, Julie K ^a   Joo, Jihoon E ^c   Jung, Chol Hee ^c   Makalic, Enes ^b   Schmidt, Daniel F ^b   Park, Daniel J ^c   Chung, Jessica ^d   Ta, Anthony D ^c   Bolton, Damien M ^c   Lonie, Andrew ^c   Longano, Anthony ^e   Hopper, John L ^b   Severi, Gianluca ^a,f,g,h   Saffery, Richard ^c,i   English, Dallas R ^a,b   Southey, Melissa C ^c   Giles, Graham G ^a,b   more..

^a CANCER EPIDEMIOLOGY CENTRE   (Australia)

^b UNIVERSITY OF MELBOURNE   (Australia)

^c UNIVERSITY OF MELBOURNE   (Australia)

^d UNIVERSITY OF TASMANIA   (Australia)

^e MONASH MEDICAL CENTRE   (Australia)

^f LABORATOIRE DES SCIENCES DU CLIMAT ET DE L ENVIRONNEMENT   (France)

^g INSTITUT GUSTAVE ROUSSY   (France)

^h Human Genetics Foundation   (Italy)

ⁱ ROYAL CHILDREN'S HOSPITAL   (Australia)

Author keywords

biomarker; bladder cancer; DNA methylation; EWAS; peripheral blood; urothelial cell carcinoma

Indexed keywords

CYANOCOBALAMIN; FOLIC ACID; DNA;

ADULT; AGED; ALCOHOL CONSUMPTION; ARTICLE; BLOOD; BLOOD SAMPLING; BODY MASS; CANCER RISK; CASE CONTROL STUDY; CONTROLLED STUDY; DNA METHYLATION; FEMALE; FOLLOW UP; HUMAN; MAJOR CLINICAL STUDY; MALE; PRIORITY JOURNAL; PROMOTER REGION; PROSPECTIVE STUDY; SMOKING; SOCIAL STATUS; TRANSITIONAL CELL CARCINOMA; VITAMIN INTAKE; CARCINOMA, TRANSITIONAL CELL; CPG ISLAND; DIET; GENETICS; GENOME-WIDE ASSOCIATION STUDY; INCIDENCE; MIDDLE AGED; PATHOLOGY; RISK; RISK FACTOR; TIME FACTOR; TUMOR INVASION; UROLOGIC NEOPLASMS; VICTORIA;

ADULT; AGED; BLOOD SPECIMEN COLLECTION; CARCINOMA, TRANSITIONAL CELL; CASE-CONTROL STUDIES; CPG ISLANDS; DIET; DNA; DNA METHYLATION; FEMALE; FOLLOW-UP STUDIES; GENOME-WIDE ASSOCIATION STUDY; HUMANS; INCIDENCE; MALE; MIDDLE AGED; NEOPLASM INVASIVENESS; PROSPECTIVE STUDIES; RISK; RISK FACTORS; SMOKING; TIME FACTORS; UROLOGIC NEOPLASMS; VICTORIA;

EID: 84982920920     PISSN: 00070920     EISSN: 15321827     Source Type: Journal    
DOI: 10.1038/bjc.2016.237     Document Type: Article

References (62)

1. Aine M, Eriksson P, Liedberg F, Hoglund M, Sjodahl G (2015a) On molecular classification of bladder cancer: out of one, many. Eur Urol 68(6): 921-923.
2. Aine M, Eriksson P, Liedberg F, Sjodahl G, Hoglund M (2015b) Biological determinants of bladder cancer gene expression subtypes. Sci Rep 5: 10957.
3. Andreotti G, Karami S, Pfeiffer RM, Hurwitz L, Liao LM, Weinstein SJ, Albanes D, Virtamo J, Silverman DT, Rothman N, Moore LE (2014) LINE1 methylation levels associated with increased bladder cancer risk in pre-diagnostic blood DNA among US (PLCO) and European (ATBC) cohort study participants. Epigenetics 9(3): 404-415.
4. Antequera F, Tamame M, Villanueva JR, Santos T (1984) DNA methylation in the fungi. J Biol Chem 259(13): 8033-8036.
5. Aryee MJ, Jaffe AE, Corrada-Bravo H, Ladd-Acosta C, Feinberg AP, Hansen KD, Irizarry RA (2014) Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays. Bioinformatics 30(10): 1363-1369.
6. Besaratinia A, Cockburn M, Tommasi S (2013) Alterations of DNA methylome in human bladder cancer. Epigenetics 8(10): 1013-1022.
7. Bestor TH, Hellewell SB, Ingram VM (1984) Differentiation of two mouse cell lines is associated with hypomethylation of their genomes. Mol Cell Biol 4(9): 1800-1806.
8. Bilgrami SM, Qureshi SA, Pervez S, Abbas F (2014) Promoter hypermethylation of tumor suppressor genes correlates with tumor grade and invasiveness in patients with urothelial bladder cancer. Springer Plus 3: 178.
9. Bose M, Wu C, Pankow JS, Demerath EW, Bressler J, Fornage M, Grove ML, Mosley TH, Hicks C, North K, Kao WH, Zhang Y, Boerwinkle E, Guan W (2014) Evaluation of microarray-based DNA methylation measurement using technical replicates: the Atherosclerosis Risk In Communities (ARIC) Study. BMC Bioinformatics 15: 312.
10. Brennan K, Flanagan JM (2012a) Epigenetic epidemiology for cancer risk: harnessing germline epigenetic variation. Methods Mol Biol 863: 439-465.
11. Brennan K, Flanagan JM (2012b) Is there a link between genome-wide hypomethylation in blood and cancer risk? Cancer Prev Res (Phila) 5(12): 1345-1357.
12. Burger M, Catto JW, Dalbagni G, Grossman HB, Herr H, Karakiewicz P, Kassouf W, Kiemeney LA, La Vecchia C, Shariat S, Lotan Y (2013) Epidemiology and risk factors of urothelial bladder cancer. Eur Urol 63(2): 234-241.
13. Cash HL, Tao L, Yuan JM, Marsit CJ, Houseman EA, Xiang YB, Gao YT, Nelson HH, Kelsey KT (2012) LINE-1 hypomethylation is associated with bladder cancer risk among nonsmoking Chinese. Int J Cancer 130(5): 1151-1159.
14. Cho NY, Kim BH, Choi M, Yoo EJ, Moon KC, Cho YM, Kim D, Kang GH (2007) Hypermethylation of CpG island loci and hypomethylation of LINE-1 and Alu repeats in prostate adenocarcinoma and their relationship to clinicopathological features. J Pathol 211(3): 269-277.
15. Choi IS, Estecio MR, Nagano Y, Kim DH, White JA, Yao JC, Issa JP, Rashid A (2007) Hypomethylation of LINE-1 and Alu in well-differentiated neuroendocrine tumors (pancreatic endocrine tumors and carcinoid tumors). Mod Pathol 20(7): 802-810.
16. Chuang SC, Stolzenberg-Solomon R, Ueland PM, Vollset SE, Midttun O, Olsen A, Tjonneland A, Overvad K, Boutron-Ruault MC, Morois S, Clavel-Chapelon F, Teucher B, Kaaks R, Weikert C, Boeing H, Trichopoulou A, Benetou V, Naska A, Jenab M, Slimani N, Romieu I, Michaud DS, Palli D, Sieri S, Panico S, Sacerdote C, Tumino R, Skeie G, Duell EJ, Rodriguez L, Molina-Montes E, Huerta JM, Larranaga N, Gurrea AB, Johansen D, Manjer J, Ye W, Sund M, Peeters PH, Jeurnink S, Wareham N, Khaw KT, Crowe F, Riboli E, Bueno-de-Mesquita B, Vineis P (2011) A U-shaped relationship between plasma folate and pancreatic cancer risk in the European Prospective Investigation into Cancer and Nutrition. Eur J Cancer 47(12): 1808-1816.
17. Cordaux R, Batzer MA (2009) The impact of retrotransposons on human genome evolution. Nat Rev Genet 10(10): 691-703.
18. Du P, Zhang X, Huang CC, Jafari N, Kibbe WA, Hou L, Lin SM (2010) Comparison of Beta-value and M-value methods for quantifying methylation levels by microarray analysis. BMC Bioinformatics 11: 587.
19. Dugué PA, English DR, MacInnis RJ, Joo JE, Jung CH, Milne RL (2015) The repeatability of DNA methylation measures may also affect the power of epigenome-wide association studies. Int J Epidemiol 44(4): 1460-1461.
20. Dugué PA, English DR, MacInnis RJ, Jung CH, Bassett JK, Fitzgerald LM, Wong EM, Joo JE, Hopper JL, Southey MC, Giles GG, Milne RL (2016) Reliability of DNA methylation measures from dried blood spots and mononuclear cells using the Human Methylation 450k Bead Array. Sci Rep 26(6): 30317.
21. Fraga MF, Uriol E, Borja Diego L, Berdasco M, Esteller M, Canal MJ, Rodriguez R (2002) High-performance capillary electrophoretic method for the quantification of 5-methyl 2′-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues. Electrophoresis 23(11): 1677-1681.
22. Friso S, Choi SW, Girelli D, Mason JB, Dolnikowski GG, Bagley PJ, Olivieri O, Jacques PF, Rosenberg IH, Corrocher R, Selhub J (2002) A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status. Proc Natl Acad Sci USA 99(8): 5606-5611.
23. Gama-Sosa MA, Slagel VA, Trewyn RW, Oxenhandler R, Kuo KC, Gehrke CW, Ehrlich M (1983) The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res 11(19): 6883-6894.
24. Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5(10): R80.
25. Giles GG, English DR (2002) The Melbourne Collaborative Cohort Study. IARC Sci Publ 156: 69-70.
26. Harper KN, Peters BA, Gamble MV (2013) Batch effects and pathway analysis: two potential perils in cancer studies involving DNA methylation array analysis. Cancer Epidemiol Biomarkers Prev 22(6): 1052-1060.
27. Houseman EA, Accomando WP, Koestler DC, Christensen BC, Marsit CJ, Nelson HH, Wiencke JK, Kelsey KT (2012) DNA methylation arrays as surrogate measures of cell mixture distribution. BMC Bioinformatics 13: 86.
28. Jaffe AE, Irizarry RA (2014) Accounting for cellular heterogeneity is critical in epigenome-wide association studies. Genome Biol 15(2): R31.
29. Ji HX, Zhao Q, Pan JH, Shen WH, Chen ZW, Zhou ZS (2013) Association of BLCA-4 hypomethylation in blood leukocyte DNA and the risk of bladder cancer in a Chinese population. Pathol Oncol Res 19(2): 205-210.
30. Jones PA (2012) Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet 13(7): 484-492.
31. Joo JE, Wong EM, Baglietto L, Jung CH, Tsimiklis H, Park DJ, Wong NC, English DR, Hopper JL, Severi G, Giles GG, Southey MC (2013) The use of DNA from archival dried blood spots with the Infinium Human Methylation 450 array. BMC Biotechnol 13: 23.
32. Kitchen MO, Bryan RT, Haworth KE, Emes RD, Luscombe C, Gommersall L, Cheng KK, Zeegers MP, James ND, Devall AJ, Fryer AA, Farrell WE (2015) Methylation of HOXA9 and ISL1 predicts patient outcome in highgrade non-invasive bladder cancer. PLoS One 10(9): e0137003.
33. Kuo KC, McCune RA, Gehrke CW, Midgett R, Ehrlich M (1980) Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA. Nucleic Acids Res 8(20): 4763-4776.
34. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann Y, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette-Stamm L, Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la Bastide M, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowki J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ (2001) Initial sequencing and analysis of the human genome. Nature 409(6822): 860-921.
35. Li G, Liu Y, Yin H, Zhang X, Mo X, Tang J, Chen W (2014) E-cadherin gene promoter hypermethylation may contribute to the risk of bladder cancer among Asian populations. Gene 534(1): 48-53.
36. Lin YL, Gui SL, Ma JG (2015) Aberrant methylation of CDH11 predicts a poor outcome for patients with bladder cancer. Oncol Lett 10(2): 647-652.
37. Maksimovic J, Gordon L, Oshlack A (2012) SWAN: subset-quantile within array normalization for Illumina Infinium Human Methylation 450 Bead Chips. Genome Biol 13(6): R44.
38. Marsit CJ, Houseman EA, Christensen BC, Gagne L, Wrensch MR, Nelson HH, Wiemels J, Zheng S, Wiencke JK, Andrew AS, Schned AR, Karagas MR, Kelsey KT (2010) Identification of methylated genes associated with aggressive bladder cancer. PLoS One 5(8): e12334.
39. Mendoza-Perez J, Gu J, Herrera LA, Tannir NM, Matin SF, Karam JA, Huang M, Chang DW, Wood CG, Wu X (2015) Genomic DNA hypomethylation and risk of renal cell carcinoma: a case-control study. Clin Cancer Res 22(8): 2074-2082.
40. Moore LE, Pfeiffer RM, Poscablo C, Real FX, Kogevinas M, Silverman D, Garcia-Closas R, Chanock S, Tardon A, Serra C, Carrato A, Dosemeci M, Garcia-Closas M, Esteller M, Fraga M, Rothman N, Malats N (2008) Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish Bladder Cancer Study: a case-control study. Lancet Oncol 9(4): 359-366.
41. Naeem H, Wong NC, Chatterton Z, Hong MK, Pedersen JS, Corcoran NM, Hovens CM, Macintyre G (2014) Reducing the risk of false discovery enabling identification of biologically significant genome-wide methylation status using the Human Methylation 450 array. BMC Genomics 15: 51.
42. Price EM, Cotton AM, Penaherrera MS, McFadden DE, Kobor MS, Robinson W (2012) Different measures of 'genome-wide' DNA methylation exhibit unique properties in placental and somatic tissues. Epigenetics 7(6): 652-663.
43. Price ME, Cotton AM, Lam LL, Farre P, Emberly E, Brown CJ, Robinson WP, Kobor MS (2013) Additional annotation enhances potential for biologically-relevant analysis of the Illumina Infinium Human Methylation 450 Bead Chip array. Epigenetics Chromatin 6(1): 4.
44. R Core Team (2015) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing: Vienna, Austria. Available from http://www.R-project.org/.
45. Rager JE, Tilley SK, Tulenko SE, Smeester L, Ray PD, Yosim A, Currier JM, Ishida MC, Gonzalez-Horta Mdel C, Sanchez-Ramirez B, Ballinas-Casarrubias L, Gutierrez-Torres DS, Drobna Z, Del Razo LM, Garcia-Vargas GG, Kim WY, Zhou YH, Wright FA, Styblo M, Fry RC (2015) Identification of novel gene targets and putative regulators of arsenicassociated DNA methylation in human urothelial cells and bladder cancer. Chem Res Toxicol 28(6): 1144-1155.
46. Salas LA, Villanueva CM, Tajuddin SM, Amaral AF, Fernandez AF, Moore LE, Carrato A, Tardon A, Serra C, Garcia-Closas R, Basagana X, Rothman N, Silverman DT, Cantor KP, Kogevinas M, Real FX, Fraga MF, Malats N (2014) LINE-1 methylation in granulocyte DNA and trihalomethane exposure is associated with bladder cancer risk. Epigenetics 9(11): 1532-1539.
47. Schmid CW (1996) Alu: structure, origin, evolution, significance and function of one-tenth of human DNA. Prog Nucleic Acids Res Mol Biol 53: 283-319.
48. Schulz WA, Steinhoff C, Florl AR (2006) Methylation of endogenous human retroelements in health and disease. Current Topics Microbiol Immunol 310: 211-250.
49. Severi G, Southey MC, English DR, Jung CH, Lonie A, McLean C, Tsimiklis H, Hopper JL, Giles GG, Baglietto L (2014) Epigenome-wide methylation in DNA from peripheral blood as a marker of risk for breast cancer. Breast Cancer Res Treat 148(3): 665-673.
50. Shariat SF, Sfakianos JP, Droller MJ, Karakiewicz PI, Meryn S, Bochner BH (2010) The effect of age and gender on bladder cancer: a critical review of the literature. BJU Int 105(3): 300-308.
51. Shvetsov YB, Song MA, Cai Q, Tiirikainen M, Xiang YB, Shu XO, Yu H (2015) Intraindividual variation and short-term temporal trend in DNA methylation of human blood. Cancer Epidemiol Biomarkers Prev 24(3): 490-497.
52. Skinner HG, Gangnon RE, Litzelman K, Johnson RA, Chari ST, Petersen GM, Boardman LA (2012) Telomere length and pancreatic cancer: a casecontrol study. Cancer Epidemiol Biomarkers Prev 21(11): 2095-2100.
53. Tajuddin S, Amaral A, Fernández A, Chanock S, Silverman D, Tardón A, Carrato A, García-Closas M, Jackson B, Toraño E (2014) LINE-1 methylation in leukocyte DNA, interaction with phosphatidylethanolamine N-methyltransferase variants and bladder cancer risk. Br J Cancer 110(8): 2123-2130.
54. van Veldhoven K, Polidoro S, Baglietto L, Severi G, Sacerdote C, Panico S, Mattiello A, Palli D, Masala G, Krogh V, Agnoli C, Tumino R, Frasca G, Flower K, Curry E, Orr N, Tomczyk K, Jones ME, Ashworth A, Swerdlow A, Chadeau-Hyam M, Lund E, Garcia-Closas M, Sandanger TM, Flanagan JM, Vineis P (2015) Epigenome-wide association study reveals decreased average methylation levels years before breast cancer diagnosis. Clin Epigenetics 7(1): 67.
55. Wagner I, Capesius I (1981) Determination of 5-methylcytosine from plant DNA by high-performance liquid chromatography. Biochim Biophys Acta 654(1): 52-56.
56. Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S, Rebhan M, Schubeler D (2007) Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet 39(4): 457-466.
57. Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, Fiala E, Ehrlich M, Laird PW (2005) Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res 33(21): 6823-6836.
58. Wilhelm CS, Kelsey KT, Butler R, Plaza S, Gagne L, Zens MS, Andrew AS, Morris S, Nelson HH, Schned AR, Karagas MR, Marsit CJ (2010) Implications of LINE1 methylation for bladder cancer risk in women. Clin Cancer Res 16(5): 1682-1689.
59. Wong Doo N, Makalic E, Joo JE, Vajdic CM, Schmidt DF, Wong EM, Jung CH, Severi G, Park DJ, Chung J, Baglietto L, Prince HM, Seymour JF, Tam C, Hopper JL, English DR, Milne RL, Harrison SJ, Southey MC, Giles GG (2016) Global measures of peripheral blood-derived DNA methylation as a risk factor in the development of mature B-cell neoplasms. Epigenomics 8(1): 55-66.
60. Woo HD, Kim J (2012) Global DNA hypomethylation in peripheral blood leukocytes as a biomarker for cancer risk: a meta-analysis. PLoS One 7(4): e34615.
61. Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP (2004) A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 32(3): e38.
62. Ziller MJ, Hansen KD, Meissner A, Aryee MJ (2015) Coverage recommendations for methylation analysis by whole-genome bisulfite sequencing. Nat Methods 12(3): 230-232.