1 / 26

HTS DNA methylomics

HTS DNA methylomics. Guillermo Carbajosa. Blizard Institute of Cell and Molecular Science. Only known epigenetic modification of the DNA Well studied Correlation with expression and histone modifications (but complex) Correlation with disease/phenotypes Need only DNA (human studies)

kana
Download Presentation

HTS DNA methylomics

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. HTS DNA methylomics Guillermo Carbajosa Blizard Institute of Cell and Molecular Science

  2. Only known epigenetic modification of the DNA • Well studied • Correlation with expression and histone modifications (but complex) • Correlation with disease/phenotypes • Need only DNA (human studies) • Can be assayed by various methods: • (i) Restriction enzyme-based • (ii) Bisulfite-based • (iii) Immunoprecipitation DNA Methylation

  3. Methylation on the honeybee Nutritional Control of Reproductive Status in Honeybees via DNA Methylation R. Kucharski, J. Maleszka, S. Foret, R. Maleszka Disruption of methylation machinery causes enormous developmental disturbance Very interesting from the epigenetics point of view: castes, haploid vs diploid…

  4. Bisulfite sequencing (BS-seq) • Single CpG resolution Schones & Zhao, Nature Reviews Genetics, 2008 Cokus et al., Nature, 2008 Lister et al., Cell, 2008 >> Both in Arabidopsis

  5. Reduced Representation Bisulfite Sequencing (RRB-seq) Meissner et al., Nature, 2008

  6. MeDIP-seq Add solexa adapters Use only for qPCR to check MeDIP efficiency Size-select (~150 bp) and sequence MeDIP only Down TA*, Rakyan VK* Nature Biotech., 2008 • Human mature spermatozoa • Same sample also profiled by MeDIP-chip and BS-PCR • 34.2 million SE reads • - 12 million PE reads • Maq score ≥ 10 • 26.5 million reads mapped Can also use MBD proteins

  7. Comparison

  8. Methylome Comparative Studies

  9. Choice of the restriction enzymes Methylation non-sensitive Restriction site containing a CpG site Ad-mixture of 3:4 TaqaI/1:4 BsoBI/MspI

  10. Reads processing Sequenced Reads ACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGC GCTTTCCTACGGGCTT TCCTACGGGCTTACT GCTTTCCTACGGGCTT TCCTACGGGCTTACT TCCTACGGGCTTACT GCTTTCCTACGGGCTT TCCTACGGGCTTACT TCCTACGGGCTTACT Map to the Genome Minimum coverage of 5 reads

  11. Reads filtering (nitty gritty) Sequenced Reads Filter Adapters Trim the ends Map to the Genome Mapping quality Mapped to in silico digestion Minimum coverage of 5 reads

  12. Why trimming? • The regions attached to the adapters do not bisulfite convert properly • When you call methylation for that region you will get a lot of wrongly called CpG sites ACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGC CGGGTTTATTTGCTTTTTTATGGGC TGGGTTTATTTGCTTTTTTATGGGC TGGGTTTATTTGCTTTCCTATGGGC CGGGCTTATTTGCTTTCCTATGGGC Please cut along the dashed line

  13. In silico digestion filtering Filter this

  14. Reads processing (Queen Muscle)

  15. All tissues

  16. CpG sites coverage (5x) Total number of CpGs in the Bee Genome: 8,774,868

  17. Methylation call ACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGCTTTCCTACGGGCTTACTTGC CGGGTTTATTTGCTTTTTTATGGGC TGGGTTTATTTGCTTTTTTATGGGC TGGGTTTATTTGCTTTCCTATGGGC CGGGCTTATTTGCTTTCCTATGGGC CGGGCTTATTTGCTTTCCTATGGGC 3/5 0/5 60% methylated 0% methylated

  18. Low CpG genes are more methylated 50 40 30 Methylationpercentage 20 10 0 First exon Middle exon Last exon Canonicalgene

  19. Consistent with B-Seq 50 40 30 Methylation percentage 20 10 0 First exon Middle exon Last exon Canonical gene

  20. Worker larvae is more methylated than queen larvae No difference in muscle

  21. Methylation correlates with gene expression

  22. DMRs Differentially methylated regions (i.e regions that are methylated only in some tissues) Tissue1 GCTTTCCTACMGGGCTT Tissue2 GCTTTCCTACGGGCTT

  23. DMR calling Unmethylated Methylated

  24. Chi-square approach   meth               unmeth sample1        0                  5 sample2        0                  10 sample3        4                  1 sample4        0                  15 sample5        0                  5 sample6        3                  2 sample7        0                  5 sample8        9                  1 meth               unmeth sample1        1.25               3.75 sample2        2.5                7.5 sample3        1.25               3.75 … Chi-square Do this for all the possible comparison among tissues… Bonferroni correction

  25. DMR between Worker and Queen larvae Bonferroni corrected p-value 2.979585e-08

  26. Acknowledgements Queen Mary University, London Dr Vardhman Rakyan Prof David Van Heel Dr Graham Heap Nicholas Bockett Richard Dobson Dr Elia Stupka The Genome Center Prof Francis Ratnieks Dr Thomas Down Dr Daniel Turner University of Sussex Gurdon Institute, Cambridge Sanger Institute, Cambridge

More Related