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Multiplexed DNA Methylation Analysis of Target Regions Using Microfluidics (Fluidigm)
Martyna Adamowicz1, Klio Maratou1, Timothy J Aitman2,3
1MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, London, W12 0NN, UK.
Methods in Molecular Biology (Clifton, N.J.)
|December 11, 2017
Summary
We developed a new DNA methylation analysis protocol for precise, simultaneous measurement of cytosine methylation across multiple samples and genomic regions. This method offers a cost-effective, reproducible, and high-resolution alternative for validating genome-wide methylation data.
Area of Science:
- Epigenetics
- Molecular Biology
- Genomics
Background:
- Whole genome shotgun bisulfite sequencing provides genome-wide methylation profiles.
- Existing validation methods are limited to few CpG sites and samples.
- A need exists for high-throughput, cost-effective validation of DNA methylation.
Purpose of the Study:
- To develop a multiplexed DNA methylation analysis protocol.
- To enable simultaneous quantitative measurement of cytosine methylation.
- To validate genome-wide methylation data with single nucleotide resolution.
Main Methods:
- Utilized a Fluidigm microfluidic system (48.48 Access Array).
- Performed multiplexed PCR amplification on bisulfite-converted DNA (48 samples x 48 amplicons).
- Applied sample-specific tagging, library pooling, and Illumina MiSeq sequencing.
Main Results:
- Achieved simultaneous quantitative measurement of cytosine methylation.
- Enabled single nucleotide resolution across 48 PCR amplicons and 48 samples.
- Demonstrated high reproducibility, speed, and cost-effectiveness.
Conclusions:
- The developed protocol is a powerful tool for DNA methylation analysis.
- It overcomes limitations of current validation methods for genome-wide studies.
- Offers high-throughput, accurate, and affordable assessment of DNA methylation patterns.
