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Updated: Mar 8, 2026
Targeted DNA Methylation Analysis by Next-generation Sequencing
Published on: February 24, 2015
Medium throughput bisulfite sequencing for accurate detection of 5-methylcytosine and 5-hydroxymethylcytosine
Gary G Chen1, Jeffrey A Gross1, Pierre-Eric Lutz1
1Department of Psychiatry, McGill Group for Suicide Studies, Douglas Mental Health University Institute, McGill University, Montreal, 6875 boul. LaSalle, Verdun, QC, H4H 1R3, Canada.
Background:
Epigenetic modifications of DNA, such as 5-methylcytosine and 5-hydroxymethycytosine, play important roles in development and disease. Here, we present a cost-effective and versatile methodology for the analysis of DNA methylation in targeted genomic regions, which comprises multiplexed, PCR-based preparation of bisulfite DNA libraries followed by customized MiSeq sequencing.
Results:
Using bisulfite and oxidative bisulfite conversion of DNA, we have performed multiplexed targeted sequencing to analyse several kilobases of genomic DNA in up to 478 samples, and achieved high coverage data of 5-methylcytosine and 5-hydroxymethycytosine at single-base resolution. Our results demonstrate the ability of this methodology to detect all levels of cytosine modifications at greater than 100× coverage in large sample sets at low cost compared to other targeted methods.
Conclusions:
This approach can be applied to multiple settings, from candidate gene to clinical studies, and is especially useful for validation of differentially methylated or hydroxymethylated regions following whole-genome analyses.
