Abstract
Understanding the impact of DNA methylation within different disease contexts often requires accurate assessment of these modifications in a genome-wide fashion. Frequently, patient-derived tissues stored in long-term hospital tissue banks have been preserved using formalin-fixation paraffin-embedding (FFPE). While these samples can comprise valuable resources for studying disease, the fixation process ultimately compromises the DNA’s integrity and leads to degradation. Degraded DNA can complicate CpG methylome profiling using traditional techniques, particularly when performing methylation-sensitive restriction enzyme sequencing (MRE-seq), yielding high backgrounds and resulting in lowered library complexity. Here, we describe Capture MRE-seq, a new MRE-seq protocol tailored to preserving unmethylated CpG information when using samples with highly degraded DNA. The results using Capture MRE-seq correlate well (0.92) with traditional MRE-seq calls when profiling non-degraded samples, and can recover unmethylated regions in highly degraded samples when traditional MRE-seq fails, which we validate using bisulfite sequencing-based data (WGBS) as well as methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Sinsheimer RL (1955) The action of pancreatic deoxyribonuclease. II. Isomeric dinucleotides. J Biol Chem 215(2):569–583. https://doi.org/10.1016/S0021-9258(18)65979-4
Smith ZD, Meissner A (2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14:204–220. https://doi.org/10.1038/nrg3354
Ehrlich M, Wang RY (1981) 5-methylcytosine in eukaryotic DNA. Science 212(4501):1350–1357. https://doi.org/10.1126/science.6262918
Gruenbaum Y et al (1981) Methylation of CpG sequences in eukaryotic DNA. FEBS Lett 124(1):67–71. https://doi.org/10.1016/0014-5793(81)80055-5
Greenberg MVC, Bourc’his D (2019) The diverse roles of DNA methylation in mammalian development and disease. Nat Rev Mol Cell Biol 20:590–607. https://doi.org/10.1038/s41580-019-0159-6
Lister R et al (2013) Global epigenomic reconfiguration during mammalian brain development. Science 341(6146):1237905. https://doi.org/10.1126/science.1237905
Lister R et al (2009) Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 462:315–322. https://doi.org/10.1038/nature08514
Grafstrom RH et al (1985) The characteristics of DNA methylation in an in vitro DNA synthesizing system from mouse fibroblasts. Nucleic Acids Res 13(8):2827–2842. https://doi.org/10.1093/nar/13.8.2827
Ramsahoye BH et al (2000) Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. PNAS USA 97(10):5237–5242. https://doi.org/10.1073/pnas.97.10.5237
Ziller MJ et al (2011) Genomic distribution and inter-sample variation of non-CpG methylation across human cell types. PLoS Genet 7(12):e1002389. https://doi.org/10.1371/journal.pgen.1002389
Riggs AD (1975) X inactivation, differentiation, and DNA methylation. Cytogenet Cell Genet 14(1):9–25. https://doi.org/10.1159/000130315
Patrat C et al (2020) X chromosome inactivation in human development. Development 147(1):dev183095. https://doi.org/10.1242/dev.183095
Jones PA, Taylor SM (1980) Cellular differentiation, cytidine analogs and DNA methylation. Cell 20(1):85–93. https://doi.org/10.1016/0092-8674(80)90237-8
Parry A et al (2021) Active turnover of DNA methylation during cell fate decisions. Nat Rev Genet 22:59–66. https://doi.org/10.1038/s41576-020-00287-8
Atlasi Y, Stunnenberg HG (2017) The interplay of epigenetic marks during stem cell differentiation and development. Nat Rev Genet 18:643–658. https://doi.org/10.1038/nrg.2017.57
Holiday R, Pugh JE (1975) DNA modification mechanisms and gene activity during development. Science 187(4173):226–232. https://doi.org/10.1126/science.187.4173.226
Busslinger M et al (1983) DNA methylation and the regulation of globin gene expression. Cell 31(1):197–206. https://doi.org/10.1016/0092-8674(83)90150-2
Yin Y et al (2017) Impact of cytosine methylation on DNA binding specificities of human transcription factors. Science 356(6337):eaaj2239. https://doi.org/10.1126/science.aaj2239
Schwartz D, Dennis E (1986) Transposase activity of the Ac controlling element in maize is regulated by its degree of methylation. Mol Gen Genet 205(3):476–482. https://doi.org/10.1007/BF00338085
Deniz O et al (2019) Regulation of transposable elements by DNA modification. Nat Rev Genet 20:417–431. https://doi.org/10.1038/s41576-019-0106-6
Feinberg AP, Vogelstein B (1983) Hypomethylation distinguishes some human cancers from their normal counterparts. Nature 301:89–92. https://doi.org/10.1038/301089a0
Gama-Sosa MA et al (1983) The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res 11(19):6883–6894. https://doi.org/10.1093/nar/11.19.6883
Greger V et al (1989) Epigenetic changes may contribute to the formation of spontaneous regression of retinoblastoma. Hum Genet 83(2):155–158. https://doi.org/10.1007/BF00286709
Sakai T et al (1991) Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene. Am J Hum Genet 48(5):880–888
Herman JG et al (1994) Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. PNAS USA 91(21):9700–9704. https://doi.org/10.1073/pnas.91.21.9700
Baylin SB, Jones PA (2011) A decade of exploring the cancer epigenome – biological and translational implications. Nat Rev Cancer 11:726–734. https://doi.org/10.1038/nrc3130
Klutstein M et al (2016) DNA methylation in cancer and aging. Cancer Res 76(12):3446–3450. https://doi.org/10.1158/0008-5472.CAN-15-3278
Weber M et al (2005) Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37(8):853–862. https://doi.org/10.1038/ng1598
Taiwo O et al (2012) Methylome analysis using MeDIP-seq with low DNA concentrations. Nat Protoc 7(4):612–636. https://doi.org/10.1038/nprot.2012.012
Maunakea AK et al (2010) Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature 466:253–257. https://doi.org/10.1038/nature09165
Li D et al (2015) Combining MeDIP-seq and MRE-seq to investigate genome-wide CpG methylation. Methods 72:29–40. https://doi.org/10.1016/j.ymeth.2014.10.032
Xing X et al (2018) Comprehensive whole DNA methylome analysis by integrating MeDIP-seq and MRE-seq. In: Tost J (ed) DNA methylation protocols, Methods Mol Biol, vol 1708. Humana Press, New York. https://doi.org/10.1007/978-1-4939-7481-8_12
Srinivasan M et al (2002) Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol 161(6):1961–1971. https://doi.org/10.1016/S0002-9440(10)64472-0
Dietrich D et al (2013) Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition. PLoS One 8(10):e77771. https://doi.org/10.1371/journal.pone.0077771
Guyard A et al (2017) DNA degrades during storage in formalin-fixed and paraffin-embedded tissue blocks. Virchows Arch 471(4):491–500. https://doi.org/10.1007/s00428-017-2213-0
ENCODE Project Consortium (2012) An integrated encyclopedia of DNA elements in the human genome. Nature 489:57–74. https://doi.org/10.1038/nature11247
Andrews S (2010) FastQC: a quality control tool for high throughput sequence data. Available via http://www.bioinformatics.babraham.ac.uk/projects/fastqc. Accessed 5 Sept 2018
Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17(1):10–12. https://doi.org/10.14806/ej.17.1.200
Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25(14):1754–1760. https://doi.org/10.1093/bioinformatics/btp324
Li H et al (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. https://doi.org/10.1093/bioinformatics/btp352
Zhou X et al (2011) The human epigenome browser at Washington University. Nat Methods 8:989–990. https://doi.org/10.1038/nmeth.1772
Li D et al (2019) WashU epigenome browser update 2019. Nucleic Acids Res 47(W1):W158–W165. https://doi.org/10.1093/nar/gkz348
Wang X et al (2016) Epigenomic analysis of chromatin organization and DNA methylation. In: Wong K (ed) Computational biology and bioinformatics, Gene Regulation. CRC Press, Boca Raton
Acknowledgments
We would like to thank Jess Hoisington-Lopez and Maria Lynn Jaeger from the Edison Family Center for Genome Sciences and Systems Biology at Washington University for assisting in sequencing our libraries. We would also like to thank Dr. Mark Watson and Dr. Ramaswamy Govindan for generously providing the FFPE block used in this study.
Availability
An epigenomic data hub has been created to display all data used in this study and is available at https://epigenomegateway.wustl.edu/browser/ with the accompanying session bundle ID: 5d37dcb0-ed9a-11ea-a6fa-9fc16c0f334f.
Accession Numbers
Sequencing data for GM12878 traditional MRE-seq, Capture MRE-seq, 1-min sonicated Capture MRE-seq, 10-min sonicated MRE-seq, MeDIP-seq, Lung FFPE Capture MRE-seq, and MeDIP-seq have been deposited in Gene Expression Omnibus under the BioProject ID “PRJNA656241.”
Conflict of Interest
The authors declare no conflicts of interest.
Funding
This work was supported by the National Institutes of Health [5R01HG007175 to T.W., U24ES026699 to T.W., U01HG009391 to T.W.] and the American Cancer Society Research Scholar Grant [RSG-14-049-01-DMC to T.W.].
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Xing, X., Karlow, J.A., Li, D., Jang, H.S., Lee, H.J., Wang, T. (2023). Capture Methylation-Sensitive Restriction Enzyme Sequencing (Capture MRE-Seq) for Methylation Analysis of Highly Degraded DNA Samples. In: Myers, M.B., Schandl, C.A. (eds) Clinical Applications of Nucleic Acid Amplification. Methods in Molecular Biology, vol 2621. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2950-5_6
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2950-5_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2949-9
Online ISBN: 978-1-0716-2950-5
eBook Packages: Springer Protocols