Encyclopedia of Metagenomics

Living Edition
| Editors: Karen E. Nelson

DNA Methylation Analysis by Pyrosequencing

Living reference work entry
DOI: https://doi.org/10.1007/978-1-4614-6418-1_799-1



Pyrosequencing is a sequencing-by-synthesis method that quantitatively monitors the real-time incorporation of nucleotides using an enzymatic conversion of pyrophosphate into a proportional light signal. Quantitative measures are crucial for applications such as the analysis of DNA methylation patterns, which are intensively studied in various developmental and pathological contexts as well as for bacterial identification and determination of allelic imbalance.


While Sanger sequencing has been the “gold standard” for the identification of sequence variants for a long time, pyrosequencing with its improved ability for quantification, decreased limit of detection and accelerated workflow leading to a shorter time to results, has become a valuable alternative notably for many clinical and diagnostic applications. Pyrosequencing is a sequencing-by-synthesis method, where nucleotides are incorporated complementary to a...


Angelman Syndrome Human Microbiome Project Imprint Control Region Pyrosequencing Assay Conventional Sanger Sequencing 
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  1. Casadesus J, Low D. Epigenetic gene regulation in the bacterial world. Microbiol Mol Biol Rev. 2006;70:830–56.PubMedCentralPubMedCrossRefGoogle Scholar
  2. Cortessis VK, Thomas DC, Levine AJ, Breton CV, Mack TM, Siegmund KD, et al. Environmental epigenetics: prospects for studying epigenetic mediation of exposure-response relationships. Hum Genet. 2012;131:1565–89.PubMedCentralPubMedCrossRefGoogle Scholar
  3. Davis BM, Chao MC, Waldor MK. Entering the era of bacterial epigenomics with single molecule real time DNA sequencing. Curr Opin Microbiol. 2013;16:192–8.PubMedCentralPubMedCrossRefGoogle Scholar
  4. Dejeux E, Audard V, Cavard C, Gut IG, Terris B, Tost J. Rapid identification of promoter hypermethylation in hepatocellular carcinoma by pyrosequencing of etiologically homogeneous sample pools. J Mol Diagn. 2007;9:510–20.PubMedCentralPubMedCrossRefGoogle Scholar
  5. Dupont JM, Tost J, Jammes H, Gut IG. De novo quantitative bisulfite sequencing using the pyrosequencing technology. Anal Biochem. 2004;333:119–27.PubMedCrossRefGoogle Scholar
  6. How Kit A, Nielsen HM, Tost J. DNA methylation based biomarkers: practical considerations and applications. Biochimie. 2012;94:2314–37.PubMedCrossRefGoogle Scholar
  7. Karimi M, Johansson S, Ekström TJ. Using LUMA: a Luminometric-based assay for global DNA-methylation. Epigenetics. 2006;1:45–8.PubMedCrossRefGoogle Scholar
  8. Kristensen LS, Treppendahl MB, Asmar F, Girkov MS, Nielsen HM, Kjeldsen TE, et al. Investigation of MGMT and DAPK1 methylation patterns in diffuse large B-cell lymphoma using allelic MSP-pyrosequencing. Sci Rep. 2013;3.Google Scholar
  9. Madi T, Balamurugan K, Bombardi R, Duncan G, McCord B. The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing. Electrophoresis. 2012;33:1736–45.PubMedCrossRefGoogle Scholar
  10. Marsh S, editor. Pyrosequencing protocols, methods in molecular biology vol 373. Totowa: Humana Press; 2007.Google Scholar
  11. Ogino S, Kawasaki T, Brahmandam M, Yan L, Cantor M, Namgyal C, Mino-Kenudson M, Lauwers GY, Loda M, Fuchs CS. Sensitive sequencing method for KRAS mutation detection by pyrosequencing. J Mol Diagn. 2005;7:413–21.PubMedCentralPubMedCrossRefGoogle Scholar
  12. Paliwal A, Vaissière T, Herceg Z. Quantitative detection of DNA methylation states in minute amounts of DNA from body fluids. Methods. 2010;52:242–47.PubMedCrossRefGoogle Scholar
  13. Petrosino JF, Highlander S, Luna RA, Gibbs RA, Versalovic J. Metagenomic pyrosequencing and microbial identification. Clin Chem. 2009;55:856–66.PubMedCentralPubMedCrossRefGoogle Scholar
  14. Shaw RJ, Akufo-Tetteh EK, Risk JM, Field JK, Liloglou T. Methylation enrichment pyrosequencing: combining the specificity of MSP with validation by pyrosequencing. Nucleic Acids Res. 2006;34:e78.PubMedCentralPubMedCrossRefGoogle Scholar
  15. Tost J. DNA methylation: an introduction to the biology and the disease-associated changes of a promising biomarker. Mol Biotechnol. 2009;44:71–81.CrossRefGoogle Scholar
  16. Tost J, Gut IG. DNA methylation analysis by pyrosequencing. Nat Protoc. 2007;2:2265–75.PubMedCrossRefGoogle Scholar
  17. Tost J, Elabdalaoui H, Gut IG. Serial pyrosequencing for quantitative DNA methylation analysis. Biotechniques. 2006;40:721–6.PubMedCrossRefGoogle Scholar
  18. Ugolotti E, Vanni I, Raso A, Benzi F, Malnati M, Biassoni R. Human leukocyte antigen–B (-Bw6/-Bw4 I80, T80) and human leukocyte antigen–C (-C1/-C2) subgrouping using pyrosequence analysis. Hum Immunol. 2011;72:859–68.PubMedCrossRefGoogle Scholar
  19. Wong H-L, Byun H-M, Kwan J, Campan M, Ingles S, Laird P, et al. Rapid and quantitative method of allele-specific DNA methylation analysis. Biotechniques. 2006;41:734–9.PubMedCrossRefGoogle Scholar
  20. Yang AS, Estécio MRH, Doshi K, Kondo Y, Tajara EH, Issa J-PJ. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. 2004;32:e38.PubMedCentralPubMedCrossRefGoogle Scholar
  21. Yang B, Wagner J, Yao T, Damaschke N, Jarrard DF. Pyrosequencing for the rapid and efficient quantification of allele-specific expression. Epigenetics. 2013;8:1039–42.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Laboratory for Epigenetics and EnvironmentCentre National de Génotypage, CEA- Institut de GénomiqueEvryFrance
  2. 2.Laboratory for Epigenetics and Environnement, Centre National de GénotypageCEA-Institut de GénomiqueEvry CedexFrance