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Selective Capture of 5-hydroxymethylcytosine from Genomic DNA
Published on: October 5, 2012
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Focused, high accuracy 5-methylcytosine quantitation with base resolution by benchtop next-generation sequencing
Epigenetics & Chromatin
|November 28, 2013
Summary
BiSulfite Amplicon Sequencing (BSAS) enables targeted DNA methylation analysis with high accuracy and low input. This method provides precise, quantitative assessment of CpG methylation for focused genomic studies.
Area of Science:
- Epigenetics
- Molecular Biology
- Genomics
Background:
- Growing interest in epigenetic modifications' role in health and disease.
- Need for targeted DNA methylation analysis beyond whole-genome methods.
- Development of BiSulfite Amplicon Sequencing (BSAS) for focused analysis.
Purpose of the Study:
- To introduce and validate BiSulfite Amplicon Sequencing (BSAS) for hypothesis-driven DNA methylation analysis.
- To enable targeted methylation analysis for specific genes or genomic regions.
- To provide a method for confirming genome-wide studies.
Main Methods:
- Utilizes PCR enrichment of bisulfite-converted DNA from targeted regions.
- Employs transposome-mediated library construction for rapid library generation from low input (1 ng).
- Sequencing performed on Illumina MiSeq, achieving high depth (>1,000×) for precise quantification.
- Dual indexing allows simultaneous analysis of up to 96 samples.
Main Results:
- BSAS provides quantitatively precise and accurate DNA methylation level assessment with base specificity.
- Demonstrates superior quantitative accuracy compared to existing Sanger sequencing methods.
- Enables analysis of up to 96 samples simultaneously through dual indexing.
Conclusions:
- BSAS is applicable to any genomic region and DNA source (tissue, cell culture).
- Offers a novel validation approach for rapid, highly quantitative absolute CpG methylation analysis.
- Facilitates high-throughput, targeted genomic region methylation analysis.
