Fine Mapping Regulatory Variants by Characterizing Native CpG Methylation with Nanopore Long-Read Sequencing

Area of Science:

• Epigenetics and Genomics
• Molecular Biology
• Bioinformatics

Background:

• 5-methylcytosine (5mC) is a key epigenetic modification on CpG sites.
• Short-read sequencing methods for 5mC detection face limitations like PCR bias and loss of allele information.
• Existing techniques struggle with long-range co-methylation analysis and variant-specific methylation detection.

Purpose of the Study:

• To characterize human methylome profiling using nanopore long-read sequencing.
• To demonstrate the potential of long-read sequencing for long-range co-methylation analysis with native modification calling.
• To investigate allele-specific methylation and its correlation with chromatin accessibility in the human prostate genome.

Main Methods:

• Utilized nanopore adaptive sampling sequencing targeting human CpG islands.
• Applied linkage disequilibrium (LD) R² for co-methylation analysis and identified methylation haplotype blocks (MHBs).
• Performed long-read based phasing and compared 5mCG signals to identify allele-specific methylation, integrating ATAC-seq data.

Main Results:

• Identified numerous MHBs across cell lines, with a small fraction showing long-distance co-methylation (≥1,000bp).
• Found differential 5mC methylation patterns around transcription factor binding sites (e.g., reduced at CTCF/KLF5, increased at FOXE1/ZNF354A).
• Discovered 6,390 haplotype-specific methylated regions in 22Rv1 cells, with negative correlation between methylation and chromatin accessibility.

Conclusions:

• Nanopore long-read sequencing provides native methylome profiling, retaining crucial haplotype information.
• This method enables robust long-range co-methylation analysis and identification of allele-specific methylation.
• The findings offer a novel approach to understanding genome regulation, particularly in the context of the human prostate genome.