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Table of Contents
Intro
Epigenetics Methods
Copyright
Contents
Contributors
Preface
Part One: Overview
Chapter One: Epigenetics methods in biological research
1. Introduction
2. DNA methylation methods: Gene- or locus-specific methylation analyses
3. DNA methylation methods: Global DNA methylation and methylomic analyses
4. DNA methylation methods: Additional technologies
5. Histone modification methods
6. Epigenetic methods for evaluating chromatin higher order
7. Evaluation of chromosomal proteins
8. Assessment of noncoding RNA as an epigenetic modulation
9. Additional epigenetics methods
10. Future directions in epigenetics methodology
11. Conclusions
References
Part Two: DNA methylation methods: Gene- or locus-specific methylation analyses
Chapter Two: Principles of bi-sulfite conversion of DNA and methylation-specific PCR (MSP) in biological research
1. Introduction
2. Histone methylation and its associated enzymes
2.1. Biological relevance of histone methylation-regulating enzymes
3. DNA methylation and its biological importance
4. Linking DNA methylation and histone lysine methylation
5. Detecting the genome-wide patterns of DNA methylation
6. DNA methylation profiling using bi-sulfite conversion
6.1. Major factors influencing the bi-sulfite conversion process
6.2. A modified method for bi-sulfite conversion
7. Methylation specific PCR (MS-PCR)
7.1. Analysis of MS-PCR data to quantify methylation
8. Application of MS-PCR in biological research
9. Concluding remarks
References
Chapter Three: Analysis of DNA methylation using pyrosequencing
1. Introduction
2. Principles of DNA methylation analysis by pyrosequencing
3. Overview of method
3.1. Bisulfite conversion
3.2. Assay design
3.3. PCR amplification.
3.4. Selection of the biotinylated single stranded DNA pyrosequencing template
3.5. Pyrosequencing run
3.6. Data analysis
4. Limitations of pyrosequencing
5. Applications of pyrosequencing
5.1. DNA methylation analysis
5.2. Validation of high throughput assays
5.3. DNA hydroxymethylation analysis
5.4. Genetic variation analysis
5.5. Microbial and viral typing
5.6. Pharmacogenetic research
6. Conclusion
References
Chapter Four: Methylation-specific droplet digital PCR (MS-ddPCR) for detection and absolute quantification of rare methy ...
Rationale
1. Materials, equipment, and reagents
1.1. General materials
1.2. Genomic DNA extraction
1.3. gDNA quantification
1.4. Bisulfite conversion of gDNA
1.5. Assay components
1.6. Bio-Rad QX200 ddPCR system consumables
1.7. Bio-Rad QX200 ddPCR system equipment
2. Methods
2.1. Primer and probe/assay design
2.2. Assay development and optimization
2.3. Genomic DNA extraction and quantification
2.4. gDNA bisulfite conversion
2.5. Plate layout and experimental design
2.6. Sample preparation
2.7. Droplet generation and transfer to plate
2.8. Sealing the plate
2.9. PCR
2.10. Droplet reading
3. Analysis and statistics
Notes
4. Preceding methylation-specific PCR techniques
4.1. Safety considerations and standards
5. Related techniques
6. Alternative methods/procedures
7. Summary
Financial support
Conflict of interest statement
References
Part Three: DNA methylation methods: Global DNA methylation and methylomic analyses
Chapter Five: ELISA analysis of global methylation levels
1. Introduction
2. Other chemical modifications at CpG sites in DNA
3. Positive control
4. Negative control and blank
5. Calculation of the percentage of global methylation
6. Reagents.
7. Materials
8. Stages of global methylation/hydroxymethylation analysis by ELISA
9. Problems related to the quantitation of global methylation by ELISA
10. Advantages of quantitation of global methylation by ELISA
11. Final considerations
References
Chapter Six: Bisulfite PCR of repetitive genomic sequences
1. Introduction
2. DNA methylation
3. Methylation of repetitive elements
3.1. What are repetitive elements?
3.2. LINE-1 elements
3.3. Alu elements
3.4. Repetitive element methylation as surrogate marker for global methylation
4. Main assays to measure global DNA methylation level
5. Bisulfite conversion-based PCR analysis of RE methylation
5.1. Bisulfite treatment and polymerase chain reaction (PCR) amplification
5.2. Methylation-specific PCR (MSP) and its modifications
5.3. The MethyLight assay and its modifications
5.3.1. QAMA and AQAMA
5.4. Combined bisulfite restriction analysis (COBRA)
5.5. Bisulfite conversion-based PCR analysis coupled with sequencing
5.5.1. Pyrosequencing based assay (Pyroseq)
5.5.2. Bisulfite sequencing (BS) and its modifications
Bisulfite sequencing coupled with digital PCR
Bisulfite sequencing coupled with next generation sequencing
Reduced representation bisulfite sequencing (RRBS)
5.5.3. Array-based methylation analysis
The infinium human methylation BeadChip
6. Challenges associated with analysis of RE methylation by bisulfite PCR
7. Concluding remarks and future directions
References
Chapter Seven: The Illumina Infinium methylation assay for genome-wide methylation analyses
1. Introduction
2. The HumanMethylation (HM) BeadChip platform
2.1. Basic principles of measuring DNA methylation
2.2. Manufacture and decoding of HM BeadChip arrays
2.3. Experimental and raw data acquisition procedures.
2.4. Data processing procedures
2.5. The former and current versions of HM BeadChip arrays
3. Characterization of classical epigenetic phenomena in humans
3.1. Variable X chromosome inactivation
3.2. Cataloguing imprinted differentially methylated regions
4. Disease diagnosis and subtype classification by epigenomic signatures
4.1. DNA methylation profiling of patients with mutations in epigenetic regulators
4.2. Recent examples of large-scale cancer subtype classification studies using HM BeadChip arrays
5. HM BeadChip arrays in population epigenomics
5.1. Advantages and limitations of HM BeadChip in population epigenomics
5.2. Examples of population epigenomic cohort studies using HM BeadChip arrays
5.2.1. Natural experiments
5.2.2. Longitudinal birth cohorts
5.2.3. Longitudinal twin studies
5.2.4. Prenatal cohorts
5.2.5. In vitro fertilization conception cohorts
5.2.6. PACE consortium
6. Genome-wide identification of DNA methylation quantitative loci (meQTL) by HM BeadChip arrays
7. Characterizing other sources of epigenetic variation in the human population: Age and sex-bias
7.1. Epigenetic age acceleration
7.2. Sexual dimorphism
8. Sequencing-based approaches
9. Conclusions
References
Further reading
Chapter Eight: Analysis of genome-wide methylation using reduced representation bisulfite sequencing (RRBS) technology
1. Introduction
2. The development of RRBS
3. Mammalian cell differentiation and early embryonic development researches with RRBS
4. Cancer research with RRBS
5. Aging research with RRBS
6. Smoking exposure research with RRBS
7. Conclusion and future directions
Chapter Eight. References
References
Chapter Nine: Methylated DNA immunoprecipitation sequencing (MeDIP-seq): Principles and applications.
1. An overview of methylated DNA immunoprecipitation
1.1. Methylated DNA immunoprecipitation and sequencing (MeDIP-seq)
1.2. Advantages of MeDIP-seq
1.3. MeDIP-seq challenges
2. MeDIP-seq experiment design
2.1. Best uses and potential considerations
2.2. Experimental controls
3. MeDIP-seq data analysis
4. MeDIP-seq in action
4.1. Aging
4.2. Brain health
4.3. Obesity, diabetes, and cardiovascular disease
4.4. Cancer
4.5. Environmental exposure
4.6. Single cell methylation profiling and MeDIP-seq
4.7. Agriculture applications
5. Conclusion
References
Chapter Ten: Multiplex analyses using methylation sensitive restriction enzyme qPCR
1. Introduction
2. Challenges in methylation analysis of clinical samples
2.1. Analysis of tissue DNA samples
2.2. Analysis of cell-free DNA from body fluids
2.3. Methylation-sensitive restriction digestion
2.4. Preamplification strategies for highly multiplexed qPCR testing
2.5. Multiplex MSRE qPCR
2.6. Primer design for MSRE qPCR assays
2.7. Evaluating the performance of MSRE qPCR
2.8. Primer design tools
2.9. Data storage, handling, and analysis
3. Our experiences with MSRE qPCR-How we got there and what we achieved
3.1. Methylation sensitive restriction enzymes-The history of their use for genome-wide DNA methylation studies
3.2. 360-plex DNA methylation assay
3.3. Confirmation of MSRE qPCR by bisulfite based ``gold standard ́́techniques
3.4. From microarrays to high throughput qPCR
4. Summary
5. Conclusion
Chapter Ten. References
References
Chapter Eleven: Advances in whole genome methylomic sequencing
1. Background
2. Bisulfite conversion-based methods
3. The methylome beyond 5mC
4. Enzymatic deamination
5. Methodological and platform comparisons.
5.1. Comparison of post-bisulfite WGBS library preparation methods.
Epigenetics Methods
Copyright
Contents
Contributors
Preface
Part One: Overview
Chapter One: Epigenetics methods in biological research
1. Introduction
2. DNA methylation methods: Gene- or locus-specific methylation analyses
3. DNA methylation methods: Global DNA methylation and methylomic analyses
4. DNA methylation methods: Additional technologies
5. Histone modification methods
6. Epigenetic methods for evaluating chromatin higher order
7. Evaluation of chromosomal proteins
8. Assessment of noncoding RNA as an epigenetic modulation
9. Additional epigenetics methods
10. Future directions in epigenetics methodology
11. Conclusions
References
Part Two: DNA methylation methods: Gene- or locus-specific methylation analyses
Chapter Two: Principles of bi-sulfite conversion of DNA and methylation-specific PCR (MSP) in biological research
1. Introduction
2. Histone methylation and its associated enzymes
2.1. Biological relevance of histone methylation-regulating enzymes
3. DNA methylation and its biological importance
4. Linking DNA methylation and histone lysine methylation
5. Detecting the genome-wide patterns of DNA methylation
6. DNA methylation profiling using bi-sulfite conversion
6.1. Major factors influencing the bi-sulfite conversion process
6.2. A modified method for bi-sulfite conversion
7. Methylation specific PCR (MS-PCR)
7.1. Analysis of MS-PCR data to quantify methylation
8. Application of MS-PCR in biological research
9. Concluding remarks
References
Chapter Three: Analysis of DNA methylation using pyrosequencing
1. Introduction
2. Principles of DNA methylation analysis by pyrosequencing
3. Overview of method
3.1. Bisulfite conversion
3.2. Assay design
3.3. PCR amplification.
3.4. Selection of the biotinylated single stranded DNA pyrosequencing template
3.5. Pyrosequencing run
3.6. Data analysis
4. Limitations of pyrosequencing
5. Applications of pyrosequencing
5.1. DNA methylation analysis
5.2. Validation of high throughput assays
5.3. DNA hydroxymethylation analysis
5.4. Genetic variation analysis
5.5. Microbial and viral typing
5.6. Pharmacogenetic research
6. Conclusion
References
Chapter Four: Methylation-specific droplet digital PCR (MS-ddPCR) for detection and absolute quantification of rare methy ...
Rationale
1. Materials, equipment, and reagents
1.1. General materials
1.2. Genomic DNA extraction
1.3. gDNA quantification
1.4. Bisulfite conversion of gDNA
1.5. Assay components
1.6. Bio-Rad QX200 ddPCR system consumables
1.7. Bio-Rad QX200 ddPCR system equipment
2. Methods
2.1. Primer and probe/assay design
2.2. Assay development and optimization
2.3. Genomic DNA extraction and quantification
2.4. gDNA bisulfite conversion
2.5. Plate layout and experimental design
2.6. Sample preparation
2.7. Droplet generation and transfer to plate
2.8. Sealing the plate
2.9. PCR
2.10. Droplet reading
3. Analysis and statistics
Notes
4. Preceding methylation-specific PCR techniques
4.1. Safety considerations and standards
5. Related techniques
6. Alternative methods/procedures
7. Summary
Financial support
Conflict of interest statement
References
Part Three: DNA methylation methods: Global DNA methylation and methylomic analyses
Chapter Five: ELISA analysis of global methylation levels
1. Introduction
2. Other chemical modifications at CpG sites in DNA
3. Positive control
4. Negative control and blank
5. Calculation of the percentage of global methylation
6. Reagents.
7. Materials
8. Stages of global methylation/hydroxymethylation analysis by ELISA
9. Problems related to the quantitation of global methylation by ELISA
10. Advantages of quantitation of global methylation by ELISA
11. Final considerations
References
Chapter Six: Bisulfite PCR of repetitive genomic sequences
1. Introduction
2. DNA methylation
3. Methylation of repetitive elements
3.1. What are repetitive elements?
3.2. LINE-1 elements
3.3. Alu elements
3.4. Repetitive element methylation as surrogate marker for global methylation
4. Main assays to measure global DNA methylation level
5. Bisulfite conversion-based PCR analysis of RE methylation
5.1. Bisulfite treatment and polymerase chain reaction (PCR) amplification
5.2. Methylation-specific PCR (MSP) and its modifications
5.3. The MethyLight assay and its modifications
5.3.1. QAMA and AQAMA
5.4. Combined bisulfite restriction analysis (COBRA)
5.5. Bisulfite conversion-based PCR analysis coupled with sequencing
5.5.1. Pyrosequencing based assay (Pyroseq)
5.5.2. Bisulfite sequencing (BS) and its modifications
Bisulfite sequencing coupled with digital PCR
Bisulfite sequencing coupled with next generation sequencing
Reduced representation bisulfite sequencing (RRBS)
5.5.3. Array-based methylation analysis
The infinium human methylation BeadChip
6. Challenges associated with analysis of RE methylation by bisulfite PCR
7. Concluding remarks and future directions
References
Chapter Seven: The Illumina Infinium methylation assay for genome-wide methylation analyses
1. Introduction
2. The HumanMethylation (HM) BeadChip platform
2.1. Basic principles of measuring DNA methylation
2.2. Manufacture and decoding of HM BeadChip arrays
2.3. Experimental and raw data acquisition procedures.
2.4. Data processing procedures
2.5. The former and current versions of HM BeadChip arrays
3. Characterization of classical epigenetic phenomena in humans
3.1. Variable X chromosome inactivation
3.2. Cataloguing imprinted differentially methylated regions
4. Disease diagnosis and subtype classification by epigenomic signatures
4.1. DNA methylation profiling of patients with mutations in epigenetic regulators
4.2. Recent examples of large-scale cancer subtype classification studies using HM BeadChip arrays
5. HM BeadChip arrays in population epigenomics
5.1. Advantages and limitations of HM BeadChip in population epigenomics
5.2. Examples of population epigenomic cohort studies using HM BeadChip arrays
5.2.1. Natural experiments
5.2.2. Longitudinal birth cohorts
5.2.3. Longitudinal twin studies
5.2.4. Prenatal cohorts
5.2.5. In vitro fertilization conception cohorts
5.2.6. PACE consortium
6. Genome-wide identification of DNA methylation quantitative loci (meQTL) by HM BeadChip arrays
7. Characterizing other sources of epigenetic variation in the human population: Age and sex-bias
7.1. Epigenetic age acceleration
7.2. Sexual dimorphism
8. Sequencing-based approaches
9. Conclusions
References
Further reading
Chapter Eight: Analysis of genome-wide methylation using reduced representation bisulfite sequencing (RRBS) technology
1. Introduction
2. The development of RRBS
3. Mammalian cell differentiation and early embryonic development researches with RRBS
4. Cancer research with RRBS
5. Aging research with RRBS
6. Smoking exposure research with RRBS
7. Conclusion and future directions
Chapter Eight. References
References
Chapter Nine: Methylated DNA immunoprecipitation sequencing (MeDIP-seq): Principles and applications.
1. An overview of methylated DNA immunoprecipitation
1.1. Methylated DNA immunoprecipitation and sequencing (MeDIP-seq)
1.2. Advantages of MeDIP-seq
1.3. MeDIP-seq challenges
2. MeDIP-seq experiment design
2.1. Best uses and potential considerations
2.2. Experimental controls
3. MeDIP-seq data analysis
4. MeDIP-seq in action
4.1. Aging
4.2. Brain health
4.3. Obesity, diabetes, and cardiovascular disease
4.4. Cancer
4.5. Environmental exposure
4.6. Single cell methylation profiling and MeDIP-seq
4.7. Agriculture applications
5. Conclusion
References
Chapter Ten: Multiplex analyses using methylation sensitive restriction enzyme qPCR
1. Introduction
2. Challenges in methylation analysis of clinical samples
2.1. Analysis of tissue DNA samples
2.2. Analysis of cell-free DNA from body fluids
2.3. Methylation-sensitive restriction digestion
2.4. Preamplification strategies for highly multiplexed qPCR testing
2.5. Multiplex MSRE qPCR
2.6. Primer design for MSRE qPCR assays
2.7. Evaluating the performance of MSRE qPCR
2.8. Primer design tools
2.9. Data storage, handling, and analysis
3. Our experiences with MSRE qPCR-How we got there and what we achieved
3.1. Methylation sensitive restriction enzymes-The history of their use for genome-wide DNA methylation studies
3.2. 360-plex DNA methylation assay
3.3. Confirmation of MSRE qPCR by bisulfite based ``gold standard ́́techniques
3.4. From microarrays to high throughput qPCR
4. Summary
5. Conclusion
Chapter Ten. References
References
Chapter Eleven: Advances in whole genome methylomic sequencing
1. Background
2. Bisulfite conversion-based methods
3. The methylome beyond 5mC
4. Enzymatic deamination
5. Methodological and platform comparisons.
5.1. Comparison of post-bisulfite WGBS library preparation methods.