Abstract
Epigenetic modifications, such as DNA methylation, can contribute to gene regulation and chromosomal stability. There are several methods and techniques available for methylation analysis, ranging from global methylation to gene-specific targeted regions. Bisulfite conversion enables numerous methodologies to be used for downstream applications, including pyrosequencing which measures DNA methylation at an individual CpG site level. This allows specific regions of interest to be targeted for DNA methylation analysis. Designing and optimizing pyrosequencing assays correctly is vital for the interpretation of results.
Dysregulation of DNA methylation has been implicated in human diseases, with regions such as repeat elements commonly altered. Human population studies investigating these tend to use consensus sequences to target repeat elements. However, these elements have high mutational rates, particularly Alu sequences, which could lead to assay bias and masking of changes at a regional level. Therefore, it may be more beneficial to target specific repeat elements depending upon their chromosomal location, rather than analyzing overall methylation levels.
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References
Frommer M, McDonald LE, Millar DS, Collis CM, Watt F, Grigg GW, Molloy PL, Paul CL (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci 89(5):1827–1831
Clark SJ, Statham A, Stirzaker C, Molloy PL, Frommer M (2006) DNA methylation: bisulphite modification and analysis. Nat Protoc 1(5):2353–2364
Ronaghi M, Uhlén M, Nyrén P (1998) A Sequencing Method Based on Real-Time Pyrophosphate. Science 281(5375):363–365
Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W (2001) Initial sequencing and analysis of the human genome. Nature 409(6822):860–921
de Koning AJ, Gu W, Castoe TA, Batzer MA, Pollock DD (2011) Repetitive elements may comprise over two-thirds of the human genome. PLoS Genet 7(12), e1002384
Cordaux R, Batzer MA (2009) The impact of retrotransposons on human genome evolution. Nat Rev Genet 10(10):691–703
Levin HL, Moran JV (2011) Dynamic interactions between transposable elements and their hosts. Nat Rev Genet 12(9):615–627
Liu GE, Alkan C, Jiang L, Zhao S, Eichler EE (2009) Comparative analysis of Alu repeats in primate genomes. Genome Res 19(5):876–885
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Hoad, G., Harrison, K. (2015). The Design and Optimization of DNA Methylation Pyrosequencing Assays Targeting Region-Specific Repeat Elements. In: Haggarty, P., Harrison, K. (eds) Population Epigenetics. Methods in Molecular Biology, vol 1589. Humana Press, New York, NY. https://doi.org/10.1007/7651_2015_285
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DOI: https://doi.org/10.1007/7651_2015_285
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