Methylation Analysis by Sequencing on the 5500xl Genetic Analyzer
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.
Methods for DNA Methylation Analysis
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.
The 5500xl Genetic Analyzer enables accurate, ultra–high-throughput sequencing, mapping, and counting of short DNA reads and, in combination with any of the above methylation profiling strategies, can be used for comprehensive, genome-wide mapping of methylation sites.
Three genome-wide methylation profiling strategies are available for the 5500xl Genetic Analyzer. The size of the target genome and the level of resolution you require will determine which solution will best suit your needs.
Step-by-Step Guide to DNA Methylation Analysis
The three traditional methylation profiling strategies can be successfully adapted onto SOLiD® 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.
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.
User documentation for the 5500 Series Genetic Analysis Systems can be found on the SOLiD Community. Access is through the 5500 Series User Hub. Access is limited to 5500 Series Genetic Analysis Systems users who are registered on the solid community and have been authenticated through the process of requesting permission to join the 5500 Series User Hub.
The tools you need for each step in the Methylation data analysis workflow:
|Data Analysis Step||Applied Biosystems Software||3rd-Party Software***|
|1. Align reads to reference in color space|
|2. Generate quality metrics||
|3. Generate sequencing and alignment statistics||
|4. Identify Polymorphisms||
|5. Translate color space to base space|
|6. Visualize in context of annotation||
|7. Convert to SRF for publishing|
|*** Need XSQ --> csfasta/qual converter|
Targeted Methylation Analysis using Capillary Electrophoresis