Methylation mapping is inherently more complex than genome sequencing, because the methylome exhibits much more variability than the genome. While each of us has only one genome, methylomes vary with age and between tissues, are altered by environmental factors, and show aberrations in diseases. Thus, mapping a person’s methylome at different ages, in different tissue types, and in various disease states is expected to yield valuable information about the mechanisms that lead to aging and disease.

What are the methods for analyzing DNA methylation?

A variety of methods to tackle the complex question of DNA methylation have evolved over time, including bisulfite conversion, digestion with methylation-sensitive restriction enzymes, and antibody- or 5-methylcytosine binding protein–based purification of methylated DNA.

Targeted DNA methylation analysis by bisulfite conversion and amplicon sequencing enables quantitative methylation analysis at single nucleotide resolution for targets of interest. Ion AmpliSeq targeted sequencing technology on the Ion GeneStudio S5 System provides a targeted DNA methylation analysis solution with high accuracy, ease of use, and short time to answer with low DNA input.

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Three genome-wide methylation profiling strategies are available for the Ion GeneStudio S5 Series. The size of the target genome and the level of resolution you require will determine which solution will best suit your needs.

  

Affinity enrichment

Restriction enzyme

Bisulfite conversion

Summary
Antibodies and methylated-CpG binding proteins are used to pull capture and enrich methylated genomic regions for analysis
Methylation sensitive restriction enzymes used to cleave the recognition site leaving either intact methylated or unmethylated sequences for analysis
Bisulfite treatment converts unmodified cytosine to uracil, but methylated cytosine is protected and unchanged
Resolution
~250 bp
Single-base
Single-base
CpGs covered
~23 million CpGs
~2 million CpGs
>28 million CpGs
Pros
  • Cost-effective 
  • No mutations introduced
  • High sensitivity with lower costs
  • Evaluate every CpG site
Cons
  • Bias towards hypermethylated regions 
  • Inability to predict absolute methylation level
  • Regions without enzyme restriction site are not covered
  • Higher costs
  • Higher DNA input unless a targeted approach is taken 
  • DNA degradation after treatment

Table 1. Comparison of genome-wide approaches for DNA methylation profiling. Adapted from Barros-Silva D, et al. Genes 9:429 (2018).

Step-by-step guide to DNA methylation analysis

The three traditional methylation profiling strategies can be successfully adapted onto the Ion GeneStudio System for a much more complete epigenomic mapping. Depending upon your specific scientific question, you might consider the following approaches:

  • Enzymatic genomic partition
    Separate the genome into methylated and unmethylated compartments with methylation sensitive or dependent restriction enzymes
  • Bisulfite conversion
    Chemical modification of the unmethylated cytosines in the genome
  • Enrichment of methylated DNA
    Purification of methylated genomic fragments

For help choosing the best strategy for your research, please contact your field application specialist.

MethylCode Bisulfite Conversion Kit

Fast and easy way to convert unmethylated cytosines from a DNA sample into uracils while methylated cytosines remain unchanged. The modified DNA is ideal for PCR amplification for downstream analyses, including restriction endonuclease digestion, sequencing, and microarrays.

User Guide: MethylMiner Methylated DNA Enrichment Kit

Ion AmpliSeq Methylation Panel for Cancer Research

Analyze clinically relevant targets using bisulfite method for identifying specific methylation patterns within a DNA or FFPE DNA sample. With low DNA input, the panel provides high accuracy, ease of use and short time to answer.

Ordering information

See Ion AmpliSeq Methylation Panel for Cancer Research

Run sequence using Ion GeneStudio Series system. Learn more ›

Analyze sequencing output from the Ion AmpliSeq Methylation Panel for Cancer Research using Torrent Suite and Torrent Suite plugin under the name “methylation_analysis”, available from the Themo Fisher Scientific Plugin store.

Additional third-party tools are available for the methylation data analysis workflow. Reach out to your sales representative for more information.

 White Paper: A Theoretical Understanding of 2 Base Color Codes and Its Application to Annotation, Error Detection, and Error Correction

The three traditional methylation profiling strategies can be successfully adapted onto the Ion GeneStudio System for a much more complete epigenomic mapping. Depending upon your specific scientific question, you might consider the following approaches:

  • Enzymatic genomic partition
    Separate the genome into methylated and unmethylated compartments with methylation sensitive or dependent restriction enzymes
  • Bisulfite conversion
    Chemical modification of the unmethylated cytosines in the genome
  • Enrichment of methylated DNA
    Purification of methylated genomic fragments

For help choosing the best strategy for your research, please contact your field application specialist.

MethylCode Bisulfite Conversion Kit

Fast and easy way to convert unmethylated cytosines from a DNA sample into uracils while methylated cytosines remain unchanged. The modified DNA is ideal for PCR amplification for downstream analyses, including restriction endonuclease digestion, sequencing, and microarrays.

User Guide: MethylMiner Methylated DNA Enrichment Kit

Ion AmpliSeq Methylation Panel for Cancer Research

Analyze clinically relevant targets using bisulfite method for identifying specific methylation patterns within a DNA or FFPE DNA sample. With low DNA input, the panel provides high accuracy, ease of use and short time to answer.

Ordering information

See Ion AmpliSeq Methylation Panel for Cancer Research

Run sequence using Ion GeneStudio Series system. Learn more ›

Analyze sequencing output from the Ion AmpliSeq Methylation Panel for Cancer Research using Torrent Suite and Torrent Suite plugin under the name “methylation_analysis”, available from the Themo Fisher Scientific Plugin store.

Additional third-party tools are available for the methylation data analysis workflow. Reach out to your sales representative for more information.

 White Paper: A Theoretical Understanding of 2 Base Color Codes and Its Application to Annotation, Error Detection, and Error Correction

Step-by-step guide to DNA methylation analysis

The three traditional methylation profiling strategies can be successfully adapted onto the Ion GeneStudio System for a much more complete epigenomic mapping. Depending upon your specific scientific question, you might consider the following approaches:

  • Enzymatic genomic partition
    Separate the genome into methylated and unmethylated compartments with methylation sensitive or dependent restriction enzymes
  • Bisulfite conversion
    Chemical modification of the unmethylated cytosines in the genome
  • Enrichment of methylated DNA
    Purification of methylated genomic fragments

For help choosing the best strategy for your research, please contact your field application specialist.

MethylCode Bisulfite Conversion Kit

Fast and easy way to convert unmethylated cytosines from a DNA sample into uracils while methylated cytosines remain unchanged. The modified DNA is ideal for PCR amplification for downstream analyses, including restriction endonuclease digestion, sequencing, and microarrays.

User Guide: MethylMiner Methylated DNA Enrichment Kit

Ion AmpliSeq Methylation Panel for Cancer Research

Analyze clinically relevant targets using bisulfite method for identifying specific methylation patterns within a DNA or FFPE DNA sample. With low DNA input, the panel provides high accuracy, ease of use and short time to answer.

Ordering information

See Ion AmpliSeq Methylation Panel for Cancer Research

Run sequence using Ion GeneStudio Series system. Learn more ›

Analyze sequencing output from the Ion AmpliSeq Methylation Panel for Cancer Research using Torrent Suite and Torrent Suite plugin under the name “methylation_analysis”, available from the Themo Fisher Scientific Plugin store.

Additional third-party tools are available for the methylation data analysis workflow. Reach out to your sales representative for more information.

 White Paper: A Theoretical Understanding of 2 Base Color Codes and Its Application to Annotation, Error Detection, and Error Correction

The three traditional methylation profiling strategies can be successfully adapted onto the Ion GeneStudio System for a much more complete epigenomic mapping. Depending upon your specific scientific question, you might consider the following approaches:

  • Enzymatic genomic partition
    Separate the genome into methylated and unmethylated compartments with methylation sensitive or dependent restriction enzymes
  • Bisulfite conversion
    Chemical modification of the unmethylated cytosines in the genome
  • Enrichment of methylated DNA
    Purification of methylated genomic fragments

For help choosing the best strategy for your research, please contact your field application specialist.

MethylCode Bisulfite Conversion Kit

Fast and easy way to convert unmethylated cytosines from a DNA sample into uracils while methylated cytosines remain unchanged. The modified DNA is ideal for PCR amplification for downstream analyses, including restriction endonuclease digestion, sequencing, and microarrays.

User Guide: MethylMiner Methylated DNA Enrichment Kit

Ion AmpliSeq Methylation Panel for Cancer Research

Analyze clinically relevant targets using bisulfite method for identifying specific methylation patterns within a DNA or FFPE DNA sample. With low DNA input, the panel provides high accuracy, ease of use and short time to answer.

Ordering information

See Ion AmpliSeq Methylation Panel for Cancer Research

Run sequence using Ion GeneStudio Series system. Learn more ›

Analyze sequencing output from the Ion AmpliSeq Methylation Panel for Cancer Research using Torrent Suite and Torrent Suite plugin under the name “methylation_analysis”, available from the Themo Fisher Scientific Plugin store.

Additional third-party tools are available for the methylation data analysis workflow. Reach out to your sales representative for more information.

 White Paper: A Theoretical Understanding of 2 Base Color Codes and Its Application to Annotation, Error Detection, and Error Correction