The bisulfite genomic sequencing (BGS) protocol has gained worldwide popularity as the method of choice for analyzing DNA methylation. It is this popular because it is a powerful protocol and it may be coupled with many other applications. However, users often run into a slew ofproblems, including incomplete conversion,overly degraded DNA, sub-optimal PCR amplifications, false positives, uninformative results, or altogether failed experiments. We pinpoint the reasons why these problems arise and carefully explain the critical steps toward accomplishing a successful experiment step-by-step. This protocol has worked successfully (>99.9% conversion) on as little as 100 ng of DNA derived from nearly 10-year-old DNA samples extracted from whole blood stored at -80°C and resulted in enough converted DNA for more than 50 PCRreactions. The aim of this article is to makelearning and usage of BGS easier, more efficient and standardized for all users.
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Pappas, J.J., Toulouse, A. and Bradley, W.E. (2013) The bisulfite genomic sequencing protocol. Advances in Lung Cancer, 2, 21-25. doi: 10.4236/alc.2013.21004.
 Wyatt, G.R. (1951) Recognition and estimation of 5-methylcytosine in nucleic acids. Biochemical Journal, 48, 581-584.
 Tan, M., Luo, H., Lee, S., Jin, F., Yang, J.S., Montellier, E., Buchou, T., Cheng, Z., Rousseaux, S., Rajagopal, N., Lu, Z., Ye, Z., Zhu, Q., Wysocka, J., Ye, Y., Khochbin, S., Ren, B. and Zhao, Y. (2011) Identification of 67 histone marks and histone lysine croton-ylation as a new type of histone modification. Cell, 16, 1016-1028.
 Hayatsu, H., Wataya, Y., Kai, K. and Lida, S. (1970) Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry, 9, 2858-2865.
 Frommer, M., McDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L. and Paul, C.L. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proceedings of the National Academy of Sciences of the USA, 89, 1827-1831. doi:10.1073/pnas.89.5.1827
 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, 1, 191-195.
 Li, L.C. and Dahiya, R. (2002) MethPrimer: Designing primers for methylation PCRs. Bioinformatics, 18, 1427-1431. doi:10.1093/bioinformatics/18.11.1427
 Yan, J., Zierath, J.R. and Barrès, R. (2011) Evidence for non-CpG methylation in mammals. Experimental Cell Research, 317, 2555-2561.
 Paul, C.L. and Clark, S.J. (1996) Cytosine Methylation: Quantitation by Automated Genomic Sequencing and GENESCAN Analysis. BioTechniques, 21, 126-133.
 Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D. and Baylin, S.B. (1996) Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proceedings of the National Academy of Sciences of the USA, 93, 9821-9826. doi:10.1073/pnas.93.18.9821
 Uhlmann, K., Brinckmann, A., Toliat, M.R., Ritter, H. and Nurnberg, P. (2002) Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis Electrophoresis, 23, 4072-4079.
 Wojdacz, T.K. and Dobrovic, A. (2007) Methylation-sensitive high resolution melting (MSHRM): A new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Research, 35, e41.
 Gonzalgo, M.L. and Jones, P.A. (1997) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE), Nucleic Acids Research, 25, 2529-2531.
 Ehrich, M., Nelson, M.R., Stanssens, P., Zabeau, M., Liloglou, T., Xinarianos, G., Cantor, C.R., Field, J.K. and van den Boom, D. (2005) Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry. Proceedings of the National Academy of Sciences of the USA, 102, 15785-15790. doi:10.1073/pnas.0507816102
 Adorjan, P., Distler, J., Lipscher, E., Model, F., Muller, J., Pelet, C., Braun, A., Florl, A.R., Gutig, D., Grabs, G. et al., (2002) Tumour class prediction and discovery by microarray-based DNA methylation analysis. Nucleic Acids Research, 30, e21. doi:10.1093/nar/30.5.e21