Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing...

Area of Science:

• Epigenetics and Genomics
• Molecular Biology
• Cancer Research

Background:

• DNA methylation is crucial for gene regulation, imprinting, and X chromosome inactivation.
• Aberrant CpG methylation is implicated in imprinting disorders and cancer pathogenesis.
• A need exists for quantitative, long-read, multiplexed bisulfite sequencing for targeted CpG analysis.

Purpose of the Study:

• To develop and validate single-molecule real-time bisulfite sequencing (SMRT-BS) for targeted CpG methylation analysis.
• To assess the accuracy, reproducibility, and multiplexing capability of SMRT-BS.
• To enable the analysis of longer DNA fragments for comprehensive CpG island interrogation.

Main Methods:

• Optimized bisulfite conversion and PCR for DNA fragments up to ~1.5 kb.
• Single-molecule real-time bisulfite sequencing (SMRT-BS) of overlapping amplicons.
• Validation using orthogonal bisulfite-based microarray and second-generation sequencing.
• Multiplexed analysis of CpG islands in hematological malignancy cell lines.

Main Results:

• SMRT-BS demonstrated high reproducibility (r=0.972) across various amplicon lengths.
• Validation showed strong correlation with orthogonal methods (r=0.906-0.933).
• Longer amplicons (>1.0 kb) showed slightly reduced but acceptable correlation (r=0.836-0.927).
• Multiplexing successfully identified quantitative methylation levels across 30 cell lines.

Conclusions:

• SMRT-BS is an accurate, cost-effective, and highly multiplexable targeted CpG methylation method.
• Increased sequencing depth is recommended for longer amplicons (>1.0 kb).
• SMRT-BS can interrogate ~1.5 kb amplicons, potentially covering ~91% of human CpG islands.