Sequencing based approaches for profiling dna methylation
- 1. Sequencing-Based Approaches for Profiling DNA Methylation
- 2. Introduction to DNA Methylation DNA methylation, one of the most studied epigenetic modifications, refers to the addition of a methyl group to the fifth carbon of cytosine (C) catalyzed by DNA methyltransferases (Dnmts), forming 5-methylcytosine (5mC). DNA methylation predominantly occurs in CpGs but is also found in non-CpG contexts.
- 3. Introduction to DNA Methylation Transcriptional repression Transposon inactivation X chromosome inactivation Embryonic development Genomic imprinting Alteration of chromatin structure
- 4. Next-generation sequencing (NGS) has been widely used for genome-wide DNA methylation analysis: Next-Generation Sequencing
- 6. Next-Generation Sequencing Method s Strength Weakness Resolution Quantitativ e Nature Cost WGBS Evaluate methylation state of almost every CpG sites -High cost -Substantial DNA degradation after bisulfite treatment -Cannot discriminate between 5mC and 5hmC Single base Digital High RRBS -High CGI coverage -High sensitivity -Cost-effective comparing to WGBS May exhibit a poor coverage at intergenic and distal regulatory elements -Substantial DNA degradation after bisulfite treatment -Limited to regions in proximity to enzymes’ recognition sites -Cannot discriminate between 5mC and 5hmC Single base Digital Moderat e MeDIP- seq -Cost-effective -No mutation introduced -Specific to 5mC/5hmC depending on the antibody specificity -More sensitive in regions with -Biased toward hypermethylated regions -Cannot identify individual 5mC sites -Inability to predict absolute methylation level ~100 bp Abundance Moderat e Table. A comparison among WGBS, RRBS, and MeDIP- seq.
- 7. Emerging 3rd generation sequencing technologies, including PacBio SMRT and Oxford nanopore sequencing, have been recently applied in epigenetics research. Advantages: Third-Generation Sequencing Minimal chemical modification Eliminate steps for DNA amplification Reduced input Generate longer reads Detect different types of epigenetic modifications
- 8. • PacBio SMRT allows the direct detection of base modifications by monitoring the activity of DNA polymerase during the incorporation of different fluorescently labeled nucleotides into complementary DNA strands. • The detection of diverse base modifications involves the measurement of the kinetics variation in the time between base incorporations. Third-Generation Sequencing
- 9. • Single-stranded DNA is pulled by a phage DNA polymerase through a bacterial pore in single-nucleotide steps, and the ion current through the pore is recorded. • C can be distinguished from 5mC and 5hmC based on differences in the current traces. Third-Generation Sequencing
- 10. Disadvantages: Third-Generation Sequencing Higher error rate Higher costLower throughput
- 11. THANKS FOR WATCHING! info@cd-genomics.com 1-631-275-3058 45-1 Ramsey Road, Shirley, NY 11967, USA www.cd-genomics.com
Editor's Notes
- #2: Welcome back to the CD Genomics' next generation sequencing video series. In this video, you will learn how to profile DNA methylation patterns using high-throughput sequencing approaches.
- #3: Let’s begin with the introduction to DNA methylation. DNA methylation, one of the most studied epigenetic modifications, refers to the addition of a methyl group to the fifth carbon of cytosine (C) catalyzed by DNA methyltransferases (Dnmts), forming 5-methylcytosine (5mC). DNA methylation predominantly occurs in CpGs but is also found in non-CpG contexts.
- #4: DNA methylation is heritable and has been associated with multiple cellular processes, including transcriptional repression, transposon inactivation, X chromosome inactivation, embryonic development, genomic imprinting, and the alteration of chromatin structure.
- #5: Next-generation sequencing (NGS) has widely used for genome-wide DNA methylation analysis, such as whole genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and Methylated DNA immunoprecipitation sequencing (MeDIP-Seq).
- #8: (1) Minimal chemical modification during library preparation; (2) The requirement for DNA amplification is eliminated; (3) Reduced requirement for input DNA; (4) The ability to generate longer reads; (5) The ability to directly detect different types of epigenetic modifications.