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Custom NGS panels for DNA methylation testing in applications such as clinical oncology research
Epigenetic analysis of the genome offers another layer in detection of biomarkers, in addition to genetic variations such as single nucleotide variations (SNVs), copy number variations (CNVs), and insertions and deletions (indels). One of the most investigated epigenetic biomarkers is DNA methylation, and the most studied form of methylation is 5-methylcyotsine at CpG sites (dinucleotides of cytosines followed by guanines) in DNA.
DNA methylation biomarker mapping in the genome is generally considered more challenging than sequencing DNA variant biomarkers because the epigenetic profiles typically exhibit much more variability than the DNA sequence variants. While each of us has only one genome, methylomes vary with age and between tissues, can be altered by environmental factors, and often show signature aberrations in diseases.
These DNA methylation changes can be investigated using targeted NGS, allowing mapping a person’s methylome at different ages, in different tissue types, and in various disease states. These studies are expected to yield valuable information about the mechanisms that lead to aging and disease.
What are the methods for analyzing DNA methylation using next-generation sequencing?
A variety of molecular testing methods exist to tackle the complex question of DNA methylation, and these techniques have evolved over time. Some of the most popular methods for analyzing DNA methylation include bisulfite conversion, antibody- or 5-methylcytosine binding protein–based purification of methylated DNA, and digestion with methylation-sensitive restriction enzymes. Each of these approaches can be followed by targeted next-generation sequencing (NGS) or other molecular testing methods, such as Sanger sequencing, quantitative PCR (qPCR), or microarrays. The right method for a given situation is determined by a mix of factors and pros and cons that are outlined in table 1 below.
Three DNA methylation profiling strategies available for analysis on Ion Torrent platforms are discussed in more detail below. The number of targets being investigated and the level of resolution you require will determine which solution will best suit your needs.
Targeted DNA methylation analysis using NGS by bisulfite conversion and amplicon-based targeted sequencing enables quantitative methylation analysis at single nucleotide resolution for targets of interest.
Ion AmpliSeq targeted sequencing technology on the Ion GeneStudio S5 systems and the Ion Torrent Genexus System provide tailored targeted DNA methylation analysis solutions that are accurate, fast, and easy to use. These methods are also compatible with low DNA inputs, making them especially useful in clinical research applications such as clinical oncology research where available biological specimens are often limited.
| Bisulfite conversion | Affinity enrichment | Restriction enzyme | |
| Summary | Bisulfite treatment converts unmodified cytosine to uracil, but methylated cytosine is protected and unchanged | 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 |
| Resolution | Single-base | ~250 bp | Single-base |
| CpGs covered | >28 million CpGs | ~23 million CpGs | ~2 million CpGs |
| Pros | Evaluate every CpG site with whole genome bisulfite sequencing or target only the CpG sites of interest with custom panels |
|
High sensitivity with lower costs |
| Cons |
|
|
Regions without enzyme restriction site are not covered |
Table 1. Comparison of genome-wide approaches for DNA methylation profiling. Adapted from Barros-Silva D, et al. Genes 9:429 (2018).
DNA methylation in cancer research
Step-by-step guide to DNA methylation analysis using bisulfite conversion NGS
Step 1: Design experiment
The first step in the bisulfite conversion NGS workflow for DNA methylation analysis is to design the custom panel to meet your unique clinical research needs. If you are working with human samples, you can either use the Ion AmpliSeq Methylation Panel for Cancer Research community panel that is pre-designed and pre-tested in collaboration with leading clinical cancer researchers, or you can design your own custom Ion AmpliSeq panel for DNA methylation analysis. Both of these options are available through the Ion AmpliSeq Designer tool that you can access at ampliseq.com. Log in using your Thermo Fisher account username and password, but if you do not already have an account you can create a free account in minutes.
If you have a project that is more complex, or if you are working with non-human samples, contact sales to schedule a time to discuss the details of your project with our technical teams.
Step 2: Bisulfite conversion
Next, you’ll need to perform the bisulfite conversion step. Many clinical researchers choose to use a commercial kit for this step, such as the MethylCode Bisulfite Conversion Kit. Using this kit is a 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 NGS, compatible with analysis by NGS as well as other molecular methods for downstream analyses, such as PCR amplification, restriction endonuclease digestion, and microarrays.
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Step 3: NGS library preparation and templating
The next steps in the workflow after bisulfite conversion are to prepare the samples for NGS by performing the library preparation and the templating steps.
If you’re using the one of the Ion GeneStudio S5 systems, you can either perform the library prep manually or you can automate the library prep using the Ion Chef system. After completing the library preparation, you will perform the templating and chip loading using the Ion Chef instrument.
If you are using the Ion Torrent Genexus System, the Genexus Integrated Sequencer automates both the NGS library preparation and templating steps as part of the automated workflow. Only ~10 minutes of hands-on time are required to set up the run, and then the instrument performs automated library prep, templating, sequencing, analysis, and reporting—and you can come back to view the report about 24 hours later.
Step 4: Perform next-generation sequencing
Custom Ion AmpliSeq DNA methylation panels are compatible with both the Ion GeneStudio S5 systems and the Ion Torrent Genexus System. Clinical researchers choose the GeneStudio platform for its flexibility and scalability, and its performance as a workhorse benchtop NGS system. The GeneStudio platform is well established and is supported by many research publications in laboratories worldwide.
The Genexus System is often selected by clinical researchers for its simplicity, with only one touchpoint and as little as 10 minutes of hands-on time from bisulfite-converted DNA through reporting, and for it’s rapid NGS workflow, with DNA methylation biomarker results in as little as 24 hours. This innovative NGS platform brings ease of use and automation to clinical research labs so more labs worldwide can bring NGS testing in-house.
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Step 5: Analyze data
Analyze sequencing output from the Ion AmpliSeq Methylation Panel for Cancer Research or custom Ion AmpliSeq DNA methylation panels using methylationAnalysis plugin, available from the Themo Fisher Scientific Plugin store.
The methylationAnalysis plugin performs alignment and methylation calling for amplicons on both the Watson (W) and Crick (C) strands. Each amplicon may have zero, one, or more designated CpG targets (hotspots) of interest. A summary report shows each barcode name along with the sample name, the total number of reads covering the target CpGs, and the percentage of those reads that are methylated. In addition, for each barcode, text files are generated giving the number of methylated reads, unmethylated reads and percent methylation for each amplicon.
Resources for DNA methylation analysis with NGS
For Research Use Only. Not for use in diagnostic procedures.
PMR: 005314