Genome-wide DNA methylation analysis Bi-Qing Li Key Laboratory of Systems biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
outline Background Method to distinguish 5 m. C 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
Background Method to distinguish 5 m. C 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
Background p 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. p 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. p 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), 85 -101; doi: 10. 3390/genes 1010085
Background Method to distinguish 5 m. C 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
Method to distinguish 5 m. C Biotechniques. 2010 Oct; 49(4): iii-xi
Restriction endonuclease-based analysis isoschizomer Cut unmethylated DNA Regardless of methylation neoschizomer Cut unmethylated DNA Partially affacted by Cp. G methylation Cut methylated DNA Pu: A or G, m. C: 5 -methylcytosine or 5 -hydroxymethylcytosine or N 4 -methylcytosine , These half-sites can be separated by up to 3 kb, but the optimal separation is 55 -103 base pairs Biotechniques. 2010 Oct; 49(4): iii-xi
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 Cp. Gs or between a Cp. G 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. 2010 Feb 2; 11(3): 191 -203
Bisulfite conversion of DNA Bisulfite conversion PCR Proc Natl Acad Sci U S A. 1992 Mar 1; 89(5): 1827 -31.
Bisulfite conversion of DNA Single base pair resolution, no bias DNA degradation by high temperature and low PH Incomplete conversion of unmethylated cytosine ØHigh GC density regions ØProtected by histones ØStable secondary structure elements Reduced complexity of genome, greater sequence redundancy, decreased hybridization specificity Difficult to mapping (repetitive regions) Genes 2010, 1(1), 85 -101; doi: 10. 3390/genes 1010085
Immunoprecipitation-based methods p methylated DNA immunoprecipitation (Me. DIP-seq) ØAntibody recognizes 5 mc to pull down the methylated fraction of genome ØMore sensitive to highly methylated, intermediate-Cp. G density regions p methyl-binding domain protein (MBD-seq) Ø Using the methyl-binding protein Me. CP 2 or MBD 2’s affinity for Cp. Gs Ø More sensitive to highly methylated, high-Cp. G density regions Methods. 2010 Nov; 52(3): 203 -12
Immunoprecipitation-based methods Straitforward and data relatively easier to analyze Bias associated with Cp. G density and need adjustment High(MBD) or intermediate(Me. DIP) Cp. G dense regions will be interpreted as “more methylated” than equally methylated low-Cp. G density regions Low resolution, do not yield information on individual Cp. G dinucleotides Methods. 2010 Nov; 52(3): 203 -12
Background Method to distinguish 5 m. C 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
Array-based genome wide DNA methylation analysis & restriction endonuclease Ø Digestion of one pool of genomic DNA with a methylation -sensitive restriction enzyme and mock digestion of another pool or using two different enzymes Ø Two DNA pools are amplified and labelled with different fluorescent dyes for two-color Ø Array hybridization Nat Rev Genet. 2010 Feb 2; 11(3): 191 -203
Array-based genome wide DNA methylation analysis & restriction endonuclease Comprehensive high-throughput arrays for relative methylation (CHARM) Ø Mcr. BC fractionate unmethylated DNA Ø Label methyl-depleted DNA with Cy 5 and total DNA with Cy 3 Ø Hybridized on high density arrays Cut methylated DNA Genome Res. 2008 May; 18(5): 780 -90
Array-based genome wide DNA methylation analysis & restriction endonuclease Hpa. II tiny fragment enrichment by ligation mediated PCR (HELP) Ø Digestion genomic DNA with Hpa. II and Msp. I Ø Ligation-mediated PCR for the amplification of Hpa. II or Msp. I genomic restriction fragments Cut unmethylated DNA Ø Label Hpa. II amplified with Cy 5 and Msp. I with Cy 3 Ø Array hybridization Regardless of methylation Genome Res. 2006 Aug; 16(8): 1046 -55
Array-based genome wide DNA methylation analysis & methylation immunoprecipitation Ø Enrichment of methylated fragments using 5 m. C antibody or the affinity of methyl-binding proteins Ø Input DNA and enriched DNA are labeled with different fluorescent dyes Ø Array hybridization Nat Rev Genet. 2010 Feb 2; 11(3): 191 -203
Array-based genome wide DNA methylation analysis & methylation immunoprecipitation Methylated DNA immunoprecipitation From Wikipedia, the free encyclopedia
Array-based genome wide DNA methylation analysis & bisulfite conversion ILLUMINA® EPIGENETIC ANALYSIS
Array-based genome wide DNA methylation analysis & bisulfite conversion 14, 495 protein-coding gene promoters 27, 578 Cp. G sites 110 micro. RNA gene promoters Nat Rev Genet. 2010 Feb 2; 11(3): 191 -203
Array-based genome wide DNA methylation analysis & bisulfite conversion Genome Res. 2006 Mar; 16(3): 383 -93
Array-based genome wide DNA methylation analysis & bisulfite conversion Golden. Gate Bead. Array 1536 specific Cp. G site in 371 gene Golden. Gate Methylation Cancer Panel I 1505 Cp. G sites selected from 807 genes Illumina® Epigenetics Analysis Nat Rev Genet. 2010 Feb 2; 11(3): 191 -203
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
Background Method to distinguish 5 m. C 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
NGS based genome-wide DNA methylation analysis Biotechniques. 2010 Oct; 49(4): iii-xi
NGS based genome-wide DNA methylation analysis-ROCHE 454 p. Roche/454 pyrosequencing-based massively parallel bisulfite pyrosequencing Ø Include more Cp. G sites facilitating complex methylation pattern research Ø Easier and more accurately aligned to reference, especially in repetitive regions Ø Bigger chance to cover more genotype information (SNP) adjacent to cytosine Ø Relatively high sequencing cost Ø Higher error rates in calling identical bases Genes 2010, 1(1), 85 -101; doi: 10. 3390/genes 1010085
NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA Methyl-seq ~100 -350 bp Regardless of methylation Illumina Genome Analyzer II Cut unmethylated DNA Genome Res. 2009 Jun; 19(6): 1044 -56
NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA Methyl-sensitive cut counting(MSCC) The method is similar to Methyl. Seq; however, sequencing of Msp. I libraries was reported to have little effect on the measurement of methylation and was abolished to reduce costs. Genome Med. 2009 Nov 16; 1(11): 106 Nat Biotechnol. 2009 Apr; 27(4): 361 -8
NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA methyl-DNA immunoprecipitation (Me. DIP) seq Methods. 2009 Mar; 47(3): 142 -50
NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA Reduced representation bisulfite sequencing(RRBS) Illumina Genome Analyzer Nucleic Acids Research, 2005, Vol. 33, No. 18 Nat Methods. 2010 Feb; 7(2): 133 -6 Nature. 2008 Aug 7; 454(7205): 766 -70
NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA Bisulfite padlock probes(BSPPs) Nat Biotechnol. 2009 Apr; 27(4): 353 -60
NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA Bisulfite sequencing(BS-seq) Nature. 2008 Mar 13; 452(7184): 215 -9
NGS based genome-wide DNA methylation analysis-Illumina/SOLEXA Cytosine methylome sequencing (Methyl. C-seq) Cell. 2008 May 2; 133(3): 523 -36 Nature. 2009 Nov 19; 462(7271): 315 -22 Nature. 2011 Mar 3; 471(7336): 68 -73
Background Method to distinguish 5 m. C 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
Third generation sequencing based genome-wide DNA methylation analysis-Pac. Bio single-molecule, real-time sequencing (SMRT) ZMW: zero mode waveguide Nat Biotechnol. 2010 May; 28(5): 426 -8
Third generation sequencing based genome-wide DNA methylation analysis-Pac. Bio single-molecule, real-time sequencing (SMRT) Nat Methods. 2010 Jun; 7(6): 461 -5 Nat Methods. 2010 Jun; 7(6): 435 -7
Third generation sequencing based genome-wide DNA methylation analysis-Oxford Nanopore Technologies Nat Biotechnol. 2010 May; 28(5): 426 -8
Background Method to distinguish 5 m. C 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
Illumina BS-seq data manipulation ØFASTQ file format and PHRED score ØAdaptor trimming with FASTX ØQuality control with Fast. QC ØReads filter and trimming with FASTX ØReads mapping with Bismark ØBasic analysis ØAdvanced analysis and application
Illumina BS-seq data manipulation ØFASTQ file format and PHRED score ØAdaptor trimming with FASTX ØQuality control with Fast. QC ØReads filter and trimming with FASTX ØReads mapping with Bismark ØBasic analysis ØAdvanced analysis and application
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. 6 1767– 1771
Illumina BS-seq data manipulation PHRED score Nature. 2009 Nov 19; 462(7271): 315 -22 Nucleic Acids Research, 2010, Vol. 38, No. 6 1767– 1771
Illumina BS-seq data manipulation PHRED score http: //en. wikipedia. org/wiki/FASTQ_format#cite_note-Illumina_User_Guide_1. 5 -2
Illumina BS-seq data manipulation ØFASTQ file format and PHRED score ØAdaptor trimming with FASTX ØQuality control with Fast. QC ØReads filter and trimming with FASTX ØReads mapping with Bismark ØBasic analysis ØAdvanced analysis and application
Illumina BS-seq data manipulation adaptor trimming with FASTX Nature. 2009 Nov 19; 462(7271): 315 -22
Illumina BS-seq data manipulation adaptor trimming with FASTX http: //hannonlab. cshl. edu/fastx_toolkit/index. html
Illumina BS-seq data manipulation adaptor trimming with FASTX http: //hannonlab. cshl. edu/fastx_toolkit/commandline. html#fastx_clipper_usage
Illumina BS-seq data manipulation ØFASTQ file format and PHRED score ØAdaptor trimming with FASTX ØQuality control with Fast. QC ØReads filter and trimming with FASTX ØReads mapping with Bismark ØBasic analysis ØAdvanced analysis and application
Illumina BS-seq data manipulation Quality control with Fast. QC http: //www. bioinformatics. bbsrc. ac. uk/projects/fastqc/
Illumina BS-seq data manipulation Quality control with Fast. QC
Illumina BS-seq data manipulation Quality control with Fast. QC
Illumina BS-seq data manipulation ØFASTQ file format and PHRED score ØAdaptor trimming with FASTX ØQuality control with Fast. QC ØReads filter and trimming with FASTX ØReads mapping with Bismark ØBasic analysis ØAdvanced analysis and application
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-RAD 1. fastq -Q 33 | fastq_quality_filter -Q 33 -q 20 -p 80 -o OWB 1 -filt. fastq http: //hannonlab. cshl. edu/fastx_toolkit/commandline. html#fastq_quality_filter_usage
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
Illumina BS-seq data manipulation FASTQ file format and PHRED score Adaptor trimming with FASTX Quality control with Fast. QC Reads filter and trimming with FASTX Reads mapping with Bismark Basic analysis Advanced analysis and application
Illumina BS-seq data manipulation Reads mapping with Bismark
Illumina BS-seq data manipulation Reads mapping with Bismark Bioinformatics. 2011 Jun 1; 27(11): 1571 -2.
Illumina BS-seq data manipulation Reads mapping with Bismark Two computationally converted reference Bioinformatics. 2011 Jun 1; 27(11): 1571 -2.
Illumina BS-seq data manipulation Reads mapping with Bismark
Illumina BS-seq data manipulation Reads mapping with Bismark H=A, C or T
Illumina BS-seq data manipulation Reads mapping with Bismark H=A, C or T
Illumina BS-seq data manipulation Reads mapping with Bismark H=A, C or T
Illumina BS-seq data manipulation Reads mapping with Bismark
Illumina BS-seq data manipulation Reads mapping with Bismark
Illumina BS-seq data manipulation Reads mapping with Bismark chromosome position strand context m. C All C 1 468 + CG 4 4 1 469 - CG 5 6 1 470 + CG 5 5 1 471 - CG 7 7 1 7384 - CHG 6 9 1 225896 - CHH 4 16 1 771455 + CHH 5 22 1 702235 + CHG 2 12 H=A, C or T
Illumina BS-seq data manipulation FASTQ file format and PHRED score Adaptor trimming with FASTX Quality control with Fast. QC Reads filter and trimming with FASTX Reads mapping with Bismark Basic analysis Advanced analysis and application
Illumina BS-seq data manipulation Basic analysis-Reads coverage
Illumina BS-seq data manipulation Basic analysis-Reads depth
Illumina BS-seq data manipulation Basic analysis-Reads depth percentage
Illumina BS-seq data manipulation Basic analysis- Methylation level chromosome position strand context m. C All C Methylation level 1 468 + CG 4 4 100% 1 469 - CG 5 6 83. 3% 1 470 + CG 5 5 100% 1 471 - CG 7 7 100% 1 7384 - CHG 6 9 66. 7% 1 225896 - CHH 4 16 25% 1 771455 + CHH 5 22 22. 7% 1 702235 + CHG 2 12 16. 7% H=A, C or T
Illumina BS-seq data manipulation Basic analysis-Methylaion density H=A, C or T
Illumina BS-seq data manipulation FASTQ file format and PHRED score Adaptor trimming with FASTX Quality control with Fast. QC Reads filter and trimming with FASTX Reads mapping with Bismark Basic analysis Advanced analysis and application
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 ØGene expression microarray ØRNA-seq
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. 2009 Nov 19; 462(7271): 315 -22
Illumina BS-seq data manipulation Advanced analysis and application DNA methylation and gene expression Genome Res. 2010 Mar; 20(3): 320 -31.
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, mi. RNA, and histone modification …… Genome Res. 2010 Mar; 20(3): 320 -31. Nature. 2009 Nov 19; 462(7271): 315 -22
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