1. Genome-wide DNA methylation analysis
Bi-Qing Li
Key Laboratory of Systems biology,
Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences

2. outline Background Method to distinguish 5mC
Array based genome-wide DNA methylation analysis
NGS based genome-wide DNA methylation analysis
Third generation sequencing based genome-wide DNA methylation analysis
Illumina BS-seq data manipulation

3. Background
Method to distinguish 5mC
Array based genome-wide DNA methylation analysis
NGS based genome-wide DNA methylation analysis
Third generation sequencing based genome-wide DNA methylation analysis
Illumina BS-seq data manipulation

4. Background
DNA methylation is the main covalent chemical modification of DNA involved in a variety of biological processes, including embryogenesis and development, silencing of transposable elements, regulation of gene transcription and tumorigenesis and progression.
The methylation pattern of DNA is highly variable among cells types and developmental stages and influenced by disease processes and genetic factors, which brings considerable theoretical and technological challenges for its comprehensive analysis.
Recently various high-throughput approaches have been developed and applied for the genome wide analysis of DNA methylation providing single base pair resolution, quantitative DNA methylation data with genome wide coverage.
Genes 2010, 1(1), ; doi: /genes

5. Background
Method to distinguish 5mC
Array based genome-wide DNA methylation analysis
NGS based genome-wide DNA methylation analysis
Third generation sequencing based genome-wide DNA methylation analysis
Illumina BS-seq data manipulation

6. Method to distinguish 5mC
Biotechniques Oct;49(4):iii-xi

7. Restriction endonuclease-based analysis
isoschizomer
Cut unmethylated DNA
Regardless of methylation
neoschizomer
Cut unmethylated DNA
Partially affacted by CpG methylation
Cut methylated DNA
Pu: A or G, mC: 5-methylcytosine or 5-hydroxymethylcytosine or N4-methylcytosine , These half-sites can be separated by up to 3 kb, but the optimal separation is base pairs
Biotechniques Oct;49(4):iii-xi

8. Restriction endonuclease-based analysis
Methylation-sensitive restriction digestion followed by PCR across the restriction site is a very sensitive technique that is still used in some applications today.
This method is still applicable for some locus-specific studies that require linkage of DNA methylation information across multiple kilobases, either between CpGs or between a CpG and a genetic polymorphism.
Limited by providing methylation data only at the restriction enzyme recognition sites or adjacent regions
It is extremely prone to false-positive results caused by incomplete digestion for reasons other than DNA methylation.
Nat Rev Genet Feb 2;11(3):

9. Bisulfite conversion of DNA
PCR
Proc Natl Acad Sci U S A Mar 1;89(5):

10. Bisulfite conversion of DNA
Single base pair resolution, no bias
DNA degradation by high temperature and low PH
Incomplete conversion of unmethylated cytosine
High GC density regions
Protected by histones
Stable secondary structure elements
Reduced complexity of genome, greater sequence redundancy, decreased hybridization specificity
Difficult to mapping (repetitive regions)
Genes 2010, 1(1), ; doi: /genes

11. Immunoprecipitation-based methods
methylated DNA immunoprecipitation (MeDIP-seq)
Antibody recognizes 5mc to pull down the methylated fraction of genome
More sensitive to highly methylated, intermediate-CpG density regions
methyl-binding domain protein (MBD-seq)
Using the methyl-binding protein MeCP2 or MBD2’s affinity for CpGs
More sensitive to highly methylated, high-CpG density regions
Methods Nov;52(3):203-12

12. Immunoprecipitation-based methods
Straitforward and data relatively easier to analyze
Bias associated with CpG density and need adjustment
High(MBD) or intermediate(MeDIP) CpG dense regions will be interpreted as “more methylated” than equally methylated low-CpG density regions
Low resolution, do not yield information on individual CpG dinucleotides
Methods Nov;52(3):203-12

13. Background
Method to distinguish 5mC
Array based genome-wide DNA methylation analysis
NGS based genome-wide DNA methylation analysis
Third generation sequencing based genome-wide DNA methylation analysis
Illumina BS-seq data manipulation

14. Array-based genome wide DNA methylation analysis & restriction endonuclease
Digestion of one pool of genomic DNA with a methylation-sensitive restriction enzyme and mock digestion of another pool or using two different enzymes
Two DNA pools are amplified and labelled with different fluorescent dyes for two-color
Array hybridization
Nat Rev Genet Feb 2;11(3):

15. Array-based genome wide DNA methylation analysis & restriction endonuclease
Comprehensive high-throughput arrays for relative methylation (CHARM)
McrBC fractionate unmethylated DNA
Label methyl-depleted DNA with Cy5 and total DNA with Cy3
Hybridized on high density arrays
Cut methylated DNA
Genome Res May;18(5):780-90

16. Array-based genome wide DNA methylation analysis & restriction endonuclease
HpaII tiny fragment enrichment by ligation mediated
PCR (HELP)
Cut unmethylated DNA
Regardless of methylation
Digestion genomic DNA with HpaII and MspI
Ligation-mediated PCR for the amplification of HpaII or MspI genomic restriction fragments
Label HpaII amplified with Cy5 and MspI with Cy3
Array hybridization
Genome Res Aug;16(8):

17. Array-based genome wide DNA methylation analysis & methylation immunoprecipitation
Enrichment of methylated fragments using 5mC antibody or the affinity of methyl-binding proteins
Input DNA and enriched DNA are labeled with different fluorescent dyes
Array hybridization
Nat Rev Genet Feb 2;11(3):

18. Array-based genome wide DNA methylation analysis & methylation immunoprecipitation
Methylated DNA immunoprecipitation
From Wikipedia, the free encyclopedia

19. Array-based genome wide DNA methylation analysis & bisulfite conversion
ILLUMINA® EPIGENETIC ANALYSIS

20. Array-based genome wide DNA methylation analysis & bisulfite conversion
14,495 protein-coding gene promoters
27,578 CpG sites
Nat Rev Genet. 2010
Feb 2;11(3):
110 microRNA gene promoters

21. Array-based genome wide DNA methylation analysis & bisulfite conversion
Genome Res Mar;16(3):383-93

22. Array-based genome wide DNA methylation analysis & bisulfite conversion
GoldenGate BeadArray 1536 specific CpG site in 371 gene
GoldenGate Methylation Cancer Panel I 1505 CpG sites selected from 807 genes
Illumina® Epigenetics Analysis
Nat Rev Genet Feb 2;11(3):

23. Array-based genome wide DNA methylation analysis
Easy to perform such experiments
Easy to interpret data with many well-characterized software programs
Low resolution
Not easy to distinguish one repetitive element from another in a hybridization-based method
Not truly genome-wide

24. Background
Method to distinguish 5mC
Array based genome-wide DNA methylation analysis
NGS based genome-wide DNA methylation analysis
Third generation sequencing based genome-wide DNA methylation analysis
Illumina BS-seq data manipulation

25. NGS based genome-wide DNA methylation analysis
Biotechniques Oct;49(4):iii-xi

26. NGS based genome-wide DNA methylation analysis-ROCHE 454
Roche/454 pyrosequencing-based massively parallel bisulfite pyrosequencing
Include more CpG sites facilitating complex methylation pattern research
Easier and more accurately aligned to reference, especially in repetitive regions
Bigger chance to cover more genotype information (SNP) adjacent to cytosine
Relatively high sequencing cost
Higher error rates in calling identical bases
Genes 2010, 1(1), ; doi: /genes

27. NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA
Methyl-seq
~ bp
Regardless of methylation
Illumina Genome Analyzer II
Cut unmethylated DNA
Genome Res Jun;19(6):

28. NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA
Methyl-sensitive cut counting(MSCC)
The method is similar to Methyl-Seq; however, sequencing of MspI libraries was reported to have little effect on the measurement of methylation and was abolished to reduce costs.
Genome Med Nov 16;1(11):106
Nat Biotechnol Apr;27(4):361-8

29. NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA
methyl-DNA immunoprecipitation
(MeDIP) seq
Methods Mar;47(3):142-50

30. NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA
Nature Aug 7;454(7205):766-70
Nat Methods Feb;7(2):133-6
Reduced representation bisulfite sequencing(RRBS)
Illumina Genome Analyzer
Nucleic Acids Research, 2005, Vol. 33, No. 18

31. NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA
Bisulfite padlock probes(BSPPs)
Nat Biotechnol Apr;27(4):353-60

32. NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA
Bisulfite sequencing(BS-seq)
Nature Mar 13;452(7184):215-9

33. NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA
Cytosine methylome sequencing
(MethylC-seq)
Cell May 2;133(3):523-36
Nature Nov 19;462(7271):315-22
Nature Mar 3;471(7336):68-73

34. Background
Method to distinguish 5mC
Array based genome-wide DNA methylation analysis
NGS based genome-wide DNA methylation analysis
Third generation sequencing based genome-wide DNA methylation analysis
Illumina BS-seq data manipulation

35. Third generation sequencing based genome-wide DNA methylation analysis-PacBio
single-molecule, real-time sequencing (SMRT)
ZMW: zero mode waveguide
Nat Biotechnol May;28(5):426-8

36. Third generation sequencing based genome-wide DNA methylation analysis-PacBio
single-molecule, real-time sequencing (SMRT)
Nat Methods Jun;7(6):461-5
Nat Methods Jun;7(6):435-7

37. Third generation sequencing based genome-wide DNA methylation analysis-Oxford Nanopore
Oxford Nanopore Technologies
Nat Biotechnol May;28(5):426-8

38. Background
Method to distinguish 5mC
Array based genome-wide DNA methylation analysis
NGS based genome-wide DNA methylation analysis
Third generation sequencing based genome-wide DNA methylation analysis
Illumina BS-seq data manipulation

39. Illumina BS-seq data manipulation
FASTQ file format and PHRED score
Adaptor trimming with FASTX
Quality control with FastQC
Reads filter and trimming with FASTX
Reads mapping with Bismark
Basic analysis
Advanced analysis and application

40. Illumina BS-seq data manipulation
FASTQ file format and PHRED score
Adaptor trimming with FASTX
Quality control with FastQC
Reads filter and trimming with FASTX
Reads mapping with Bismark
Basic analysis
Advanced analysis and application

41. Illumina BS-seq data manipulation FASTQ file format
FASTQ has emerged as a common file format for sharing sequencing read data combining both the sequence and an associated per base quality score
Nucleic Acids Research, 2010, Vol. 38, No –1771

42. Illumina BS-seq data manipulation PHRED score
Nature Nov 19;462(7271):315-22
Nucleic Acids Research, 2010, Vol. 38, No –1771

43. Illumina BS-seq data manipulation PHRED score

44. Illumina BS-seq data manipulation
FASTQ file format and PHRED score
Adaptor trimming with FASTX
Quality control with FastQC
Reads filter and trimming with FASTX
Reads mapping with Bismark
Basic analysis
Advanced analysis and application

45. Illumina BS-seq data manipulation adaptor trimming with FASTX
Nature Nov 19;462(7271):315-22

46. Illumina BS-seq data manipulation adaptor trimming with FASTX

47. Illumina BS-seq data manipulation adaptor trimming with FASTX

48. Illumina BS-seq data manipulation
FASTQ file format and PHRED score
Adaptor trimming with FASTX
Quality control with FastQC
Reads filter and trimming with FASTX
Reads mapping with Bismark
Basic analysis
Advanced analysis and application

49. Illumina BS-seq data manipulation Quality control with FastQC

50. Illumina BS-seq data manipulation Quality control with FastQC

51. Illumina BS-seq data manipulation Quality control with FastQC

52. Illumina BS-seq data manipulation
FASTQ file format and PHRED score
Adaptor trimming with FASTX
Quality control with FastQC
Reads filter and trimming with FASTX
Reads mapping with Bismark
Basic analysis
Advanced analysis and application

53. Illumina BS-seq data manipulation Reads filter and trimming with FASTX
e.g.1 fastq_quality_filter -Q 33 -q 20 -p 100 -v -i input -o output
e.g.2 fastq_quality_filter -q 10 -p 100 -i /usr/local/data/GBS/OWB-RAD1.fastq -Q 33 | fastq_quality_filter -Q 33-q 20 -p 80 -o OWB1-filt.fastq

54. Illumina BS-seq data manipulation Reads filter and trimming with FASTX
FASTQ quality trimmer
e.g.1 fastq_quality_trimmer -t 20 -l 35 -v -i input -o output

55. Illumina BS-seq data manipulation
FASTQ file format and PHRED score
Adaptor trimming with FASTX
Quality control with FastQC
Reads filter and trimming with FASTX
Reads mapping with Bismark
Basic analysis
Advanced analysis and application

56. Illumina BS-seq data manipulation Reads mapping with Bismark

57. Illumina BS-seq data manipulation Reads mapping with Bismark
Bioinformatics Jun 1;27(11):

58. Two computationally converted reference
Illumina BS-seq data manipulation
Reads mapping with Bismark
Two computationally converted reference
Bioinformatics Jun 1;27(11):

59. Illumina BS-seq data manipulation Reads mapping with Bismark

60. Illumina BS-seq data manipulation Reads mapping with Bismark
H=A, C or T

61. Illumina BS-seq data manipulation Reads mapping with Bismark
H=A, C or T

62. Illumina BS-seq data manipulation Reads mapping with Bismark
H=A, C or T

63. Illumina BS-seq data manipulation Reads mapping with Bismark

64. Illumina BS-seq data manipulation Reads mapping with Bismark

65. Illumina BS-seq data manipulation Reads mapping with Bismark
chromosome
position
strand
context mC
All C 1
468 + CG 4
469 - 5 6
470
471 7
7384
CHG 9
225896
CHH 16
771455 22
702235 2 12
H=A, C or T

66. Illumina BS-seq data manipulation
FASTQ file format and PHRED score
Adaptor trimming with FASTX
Quality control with FastQC
Reads filter and trimming with FASTX
Reads mapping with Bismark
Basic analysis
Advanced analysis and application

67. Illumina BS-seq data manipulation Basic analysis-Reads coverage

68. Illumina BS-seq data manipulation Basic analysis-Reads depth

69. Illumina BS-seq data manipulation Basic analysis-Reads depth percentage

70. Illumina BS-seq data manipulation Basic analysis- Methylation level
chromosome
position
strand
context mC
All C
Methylation
level 1
468 + CG 4
100%
469 - 5 6
83.3%
470
471 7
7384
CHG 9
66.7%
225896
CHH 16
25%
771455 22
22.7%
702235 2 12
16.7%
H=A, C or T

71. Illumina BS-seq data manipulation Basic analysis-Methylaion density
H=A, C or T

72. Illumina BS-seq data manipulation
FASTQ file format and PHRED score
Adaptor trimming with FASTX
Quality control with FastQC
Reads filter and trimming with FASTX
Reads mapping with Bismark
Basic analysis
Advanced analysis and application

73. Illumina BS-seq data manipulation Advanced analysis and application DNA methylation and gene expression
DNA methylation is linked to gene silencing and is considered to be an important mechanism in the regulation of gene expression
Gene expression
Gene expression microarray
RNA-seq

74. Illumina BS-seq data manipulation Advanced analysis and application DNA methylation and gene expression
proximal TSS (-150 bp to +150 bp across TSS)
Promoter (1.5 kb upstream of the TSS)
Nature Nov 19;462(7271):315-22

75. Illumina BS-seq data manipulation Advanced analysis and application DNA methylation and gene expression
Genome Res Mar;20(3):

76. Illumina BS-seq data manipulation Advanced analysis and application
Differentially methylated region(DMRs) and gene expression
DNA methylation at DNA–protein interaction sites
DNA methylation, miRNA, and histone modification ……
Genome Res Mar;20(3):
Nature Nov 19;462(7271):315-22

77. Thank you!