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DNA methylation_ understanding the language of DNA 20130806

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A review of next generation DNA sequencing based methods for characterization of DNA methylation and hydroxymethylation. Presentation by Illumina.

A review of next generation DNA sequencing based methods for characterization of DNA methylation and hydroxymethylation. Presentation by Illumina.

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  • 1. © 2013 Illumina, Inc. All rights reserved. Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cBot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iSelect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa, TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Advances in Epigenetics Swati Kadam, Ph.D. Marketing Intern Aug 2013
  • 2. Cumulative papers
  • 3. Focal Points • What is Epigenetics? • Importance of Epigenetic modifications • Current methods used to map modifications • Workflows for each method • Where Illumina fits into the picture
  • 4. 4 What is Epigenetics? http://www.cashou.com/portfolio/human_body/index.html#
  • 5. 5 What is Epigenetics? http://www.cashou.com/portfolio/human_body/index.html# Total number of cells in the body ~50 trillion: 50,000,000,000,000 cells – Each with it’s own instruction manual (DNA)  ~3164.7 million bases (A, T, G, C)  ~2% of the human genome codes for protein  ~20,687 total genes (~3000 bases each)
  • 6. 6 Epigenetics: Study of heritable changes in gene expression that occur without changes in the primary DNA sequence. What is Epigenetics?
  • 7. 7 Lets eat, Grandpa! Lets eat Grandpa!
  • 8. 8 Epigenetic modifications are like punctuation marks on the DNA
  • 9. 9 Zaidi S K et al. Mol. Cell. Biol. 2010;30:4758-4766 Introduction: Epigenetic Regulation
  • 10. 10 • Methylation of Cytosines: 5mC • Hydroxymethylation of Cytosines: 5hmC • Cytosines next to Guanines: CpG sites • Many CpGs together: CpG island • Most located near TSS • CpG Shores are sites 2 kilo bases away from CpG Islands Introduction: Epigenetic Lingo CpG Island CpG siteCpG Shores 5mC 5hmC
  • 11. 11 • DNMT: DNA MethylTransferases • DNMT1, DNMT3A/3B, DNMT3L • Maintain DNA methylation mark • TET: Ten-Eleven-Translocation • TET1, TET2, TET3 • Play an important role in removing methylC (5mC) to form hydroxymethylC (5hmC) (an intermediate in demethylation). Introduction: Epigenetic Lingo ,
  • 12. 12 Full Stop Comma Pfeifer, G. P., S. Kadam, et al. (2013). "5-hydroxymethylcytosine and its potential roles in development and cancer." Epigenetics Chromatin 6(1): 10. DNA methylation (5mC) and DNA hydroxymethylation (5hmC) DNMTs DNA Methyl Transferase TETs Ten-Eleven Translocation 1/2/3 + 5mC 5hmC Proposed intermediate to DNA demethylation TETs Ten-Eleven Translocation 1/2/3 + Other Proteins
  • 13. 13 Introduction: Importance of DNA methylation Calico Cats: Female calicos have a splotched coat pattern linked to X-inactivation. DNA methylation (Full-Stop) Females
  • 14. 14 Introduction: How are 5mC or 5hmC modifications relevant? Iqbal, K., Jin, S. G., Pfeifer, G. P. & Szabo, P. E. Reprogramming of the paternal genome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc. Natl Acad. Sci. USA 108, 3642–3647 (2011) Mom-5mC Dad-5hmC
  • 15. 15 Introduction: How are 5mC or 5hmC modifications relevant? X Mutations in DNMT1: embryo dies Mutations in DNMT 3B: Genetic disorder Immunodeficiency-centromeric instability- Facial anomalies syndrome (ICF)X Brown KD, Robertson KD. DNMT1 knockout delivers a strong blow to genome stability and cell viability. Nat Genet. 2007;39(3):289–290.
  • 16. 16 Introduction: How are 5mC or 5hmC modifications relevant? Koh K. P., Yabuuchi A., Rao S., Huang Y., Cunniff K., Nardone J., et al. (2011). Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8, 200–213. doi: 10.1016/j.stem.2011.01.008. Dawlaty M. M., Breiling A., Le T., Raddatz G., Barrasa M. I., Cheng A. W., et al. (2013). Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development. Dev. Cell 24, 310–323. doi: 10.1016/j.devcel.2012.12.015. X Mutations in Tet1: viable fetus Mutations in Tet1+ Tet2: death after birth Mutated in Blood Cancers
  • 17. 17  Epigenetic modifications are punctuation marks on DNA – DNA methylation (5mC) on Cytosine  FULL STOP – DNA hydroxymethylation (5hmC) on Cytosine  COMMA  Important during growth for proper formation of organs and adult cells  Imbalance in 5mC or 5hmC leads to cancer Take Home Message 1: Epigenetic Regulation + =
  • 18. Focal Points • What is Epigenetics? • Importance of Epigenetic modifications • Current methods used to map modifications • Workflows for each method • Where Illumina fits into the picture
  • 19. 19  Techniques currently used to map 5mC and 5hmC  Differences between the techniques  Summary of workflows Mapping Epigenetic Modifications on DNA
  • 20. 20 COMPANY CONFIDENTIAL – INTERNAL USE ONLY Human: 5hmC Monocytes: ~0.06% of cytosines moDCs: ~0.10% of cytosines moMOs: ~0.11% of cytosines Human: 5mC Monocytes: ~4.8% of cytosines moDCs: ~4.6% of cytosines moMOs: ~4.5% of cytosines These modifications are like finding a needle in a haystack Mouse: 5mC ~1% of bases ~5% of cytosines ~6 x 107 in ES cells Mouse: 5hmC ~0.1% of bases ~0.5% of cytosines ~6 x 106 in ES cells
  • 21. 21 COMPANY CONFIDENTIAL – INTERNAL USE ONLY Full Stop Comma Pfeifer, G. P., S. Kadam, et al. (2013). "5-hydroxymethylcytosine and its potential roles in development and cancer." Epigenetics Chromatin 6(1): 10. DNA methylation (5mC) DNMTs DNA Methyl Transferase TETs Ten-Eleven Translocation 1/2/3 + 5mC 5hmC Proposed intermediate to DNA demethylation TETs Ten-Eleven Translocation 1/2/3 + Other Proteins
  • 22. 22 COMPANY CONFIDENTIAL – INTERNAL USE ONLY 5mC mapping technique C G G C C G G C Bisulfite Conversion C G G C C G G C C G G C C G G C U G G U U G G U BS-Seq: BiSulfite Sequencing Library Prep + Genome wide sequencing Methylated UnMethylated C G G C C G G C T G G T T G G T
  • 23. 23 COMPANY CONFIDENTIAL – INTERNAL USE ONLY 5mC mapping technique RRBS-Seq: Reduced Representation BiSulfite Sequencing MspI digestion Genomic DNA Illumina Sequencing Illumina methylated adapters BiSulfite Conversion Size Selection PCR amplification
  • 24. 24 COMPANY CONFIDENTIAL – INTERNAL USE ONLY 5mC mapping technique Me-DIP-Seq: Methylated DNA ImmunoPrecipitation Sequencing Fragmentation Size Selection Adapter Ligation Denature to ssDNA 5mC antibody Illumina Sequencing PCR amplification Enrichment of 5mC DNA
  • 25. 25 COMPANY CONFIDENTIAL – INTERNAL USE ONLY 5mC mapping technique - methyl group - GST-MBD2b - His-MBD3L1 Rauch, T.A., Li, H., Wu, X., Pfeifer, G.P., (2006). “MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeodomain-containing genes in lung cancer cells.” Cancer Res. 66: (7939-7947) MIRA-Seq: Methylated-CpG Island Recovery Assay Sequencing Fragmentation Size Selection Adapter Ligation Incubation MIRA enrichment of the 5mC DNA Genomic DNA Isolate GST- MBD2b/DNA complex Illumina Sequencing GST-bead
  • 26. 26 COMPANY CONFIDENTIAL – INTERNAL USE ONLY Reduced Restriction BiSulfite (RRBS-Seq) Methylated – DNA Immunoprecipitation (Me-DIP) BiSulfite Sequencing (BS-Seq) Library Prep + Methylated CpG Island Recovery Assay (MIRA) Bis-SNP Bismark Bison BSMAP MethylCoder DNAA 5mC methods summary
  • 27. 27 COMPANY CONFIDENTIAL – INTERNAL USE ONLY Full Stop Comma Pfeifer, G. P., S. Kadam, et al. (2013). "5-hydroxymethylcytosine and its potential roles in development and cancer." Epigenetics Chromatin 6(1): 10. DNA hydroxymethylation (5hmC) DNMTs DNA Methyl Transferase TETs Ten-Eleven Translocation 1/2/3 + 5mC 5hmC Proposed intermediate to DNA demethylation TETs Ten-Eleven Translocation 1/2/3 + Other Proteins
  • 28. 28 5hmC mapping technique Yu, M., G. C. Hon, et al. (2012). "Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine." Nat Protoc 7(12): 2159-2170. TAB-Seq: Tet-Assisted Bisulfite Sequencing
  • 29. 29 5hmC mapping technique RRHP-Seq: Reduced Representation Hydroxymethylcytosine Profiling Sequencing MspI digestion Genomic DNA Adapters hmC protection by glucosylation 5hmC 5mC MspI digestion Illumina Sequencing PCR amplification 5hmC 5mC Size selection of enriched 5hmC DNA
  • 30. 30 5hmC mapping technique HMe-DIP-Seq: HydroxyMethylated DNA ImmunoPrecipitation Sequencing Fragmentation Adapters Denature to ssDNA 5hmC antibody 5hmC 5mC Genomic DNA Illumina Sequencing PCR amplification Size selection of enriched 5hmC DNA
  • 31. 31 5hmC methods summary Reduced Representation Hydroxymethyl Profiling Sequencing (RRHP-seq) HydroxyMethylated – DNA Immunoprecipitation (HMe-DIP) Tet-Assisted Bisulfite Sequencing (TAB-Seq) Library Prep + +
  • 32. 32 Reduced Representation Hydroxymethyl Profiling Sequencing (RRHP-seq) Tet-Assisted Bisulfite Sequencing (TAB-Seq) + + HydroxyMethylated – DNA Immunoprecipitation (HMe-DIP) Library Prep Reduced Restriction BiSulfite (RRBS-Seq) Methylated – DNA Immunoprecipitation (Me-DIP) BiSulfite Sequencing (BS-Seq) + Methylated CpG Island Recovery Assay (MIRA) TruSeq Kits Nextera Kits illumina: Total Solution MiSeq RRBS-Seq/RRHP-Seq MeDIP/hMe-DIP MIRA HiSeq WGBS-Seq/TAB-Seq RRBS-Seq/RRHP-Seq MeDIP/hMe-DIP MIRA
  • 33. 33 Summary for mapping cytosine modifications CpG Island CpG site 5mC 5hmC Modification Method (example) Reagents kit Pre-prep input gDNA Cost to prepare DNA Coverage needed (Gbp) Total Cost per sample 5mC WGBS Zymo EZ DNA methylation-Lightning kit 0.5-2ug ~ $4 90 ~ $5000 RRBS Msp1 Enzyme (NEB) + Zymo EZ DNA methylation-Lightning kit 1-2ug ~ $3 3 ~ $300 Me-DIP MeDIP kit (Active Motif) 0.1-1ug ~ $40 5 ~ $300 MIRA/MBD Active Motif Methyl Collector Ultra kit (Active Motif) 0.01-1ug ~ $20 5 ~ $300 HM 450 array Illumina Infinium Array (Targeted) 0.5ug - - $240 5hmC TAB WiseGene TAB kit + Zymo EZ DNA methylation-Lightning kit 0.5ug ~ $100 90 ~ $5000 RRHP Zymo Research 3-5 ~ $300 Hme-DIP Hme-DIP kit (Active Motif) 0.1-1ug ~ $40 3-5 ~ $300 Note: An example of the kits is provided, other kits are also substitutable. Please contact manufacturer for details or recommendations Note: Values presented here are an estimate and are subject to change.
  • 34. 34 Technique Pros Cons Areas Covered Areas Missed BS  Genome wide coverage of non-CpG methylation.  Genome wide coverage of CpGs  Single -CpG resolution  GC content  Sequence complexity  Difficulty aligning BS reads to reference.  Difficulty differentiated repeat region locations in genome  Disadvantageous at C  T SNPs  High cost per CpG covered genome-wide  Cannot distinguish between 5mC and 5hmC  CpG Islands dense  CpG Islands less dense  CpGs in repeats  Non-CpG methylation  Genome-wide CpG me  None RRBS  Genome wide coverage of CpGs in Islands  Low Cost per sample  Single CpG Resolution  Msp1 cuts at specific sites  Measures 10-15% CpGs in genome  Dependent on Sequence  Cannot distinguish between 5mC and 5hmC  CpG Islands dense  CpG in repeats  Non CpG me  Genome-wide CpG me  CpG Islands w/o enzyme site MeDIP  Genome wide coverage of non-CpG methylation.  Genome wide coverage of CpGs  Independent of sequence.  Lowest cost per CpG genome-wide  Does not interact with 5hmC  Low resolution (~150bp)  Non-specific interaction  Bias towards hyper methylated regions  CpG Islands dense  CpG Islands less dense  CpGs in repeats  Non-CpG methylation  Genome-wide CpG me  None MIRA/MBD  Genome wide coverage of CpGs in dense CpG areas  Does not interact with 5hmC  Genome wide coverage of non-CpG methylation.  Less coverage of low CpG areas  Low resolution (~150bp)  Bias towards hypermethylated regions  CpG Islands dense  CpGs in repeats  Non CpG me  CpG Islands less dense  Genome wide CpG me
  • 35. 35 Reference Guides:
  • 36. Focal Points  What is Epigenetics?  Importance of Epigenetic modifications  Current methods used to map modifications  Workflows for each method  Where Illumina fits into the picture
  • 37. 37 COMPANY CONFIDENTIAL – INTERNAL USE ONLY - Identification of novel biomarkers in cancer - OPT gene 100% methylated in primary breast tumors - Kim, Myung Soon, et al. "Genome-wide identification of OTP gene as a novel methylation marker of breast cancer." Oncology reports 27.5 (2012): 1681-1688. - Identification of changes in location of 5mC and 5hmC in development - CH methylation accumulates during brain development [non-CpG methylation] - Lister, Ryan, et al. "Global Epigenomic Reconfiguration During Mammalian Brain Development." Science (New York, NY) (2013). - Changes in epigenome upon treatment with carcinogens - Non-genotoxic carcinogens causes changes in epigenome. 5hmC associated with active chromatin state. - Thomson JP, Lempiainen H, Hackett JA, et al. Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome. Genome Biol. 2012;13:R93. - Monozyotic twins studied at epigenome level - Same genome, different epigenome Where DNA methylation correlated with Autism - Wong CC, Meaburn EL, Ronald A, et al. Methylomic analysis of monozygotic twins discordant for autism spectrum disorder and related behavioural traits. Mol Psychiatry. 2013 Apr 23. doi: 10.1038/mp.2013.41. We are just beginning to scratch the surface =
  • 38. 38 Won’t you want to learn the punctuated language of your DNA to unravel the mysteries of life and disease?
  • 39. 39 Questions about presentation: – Swati Kadam, Product Marketing Intern  skadam@illumina.com – Abizar Lakdawalla, Product Management  alakdawalla@illumina.com For more information about WGBS kit: – Fraz Syed, Sr. Product Marketing  fraz.syed@epicentre.com Contact Information: