Fine mapping regulatory variants by characterizing native CpG methylation with nanopore long-read sequencing

5-Methylcytosine (5mC) is the most common DNA modification in the human genome. Bisulfite conversion combined with short-read sequencing captures this modification at single-nucleotide resolution but introduces PCR duplication bias and limits co-methylation analysis between distant cytosines. To resolve these limitations, we used nanopore long-read sequencing to profile human methylation and performed long-range co-methylation analysis with native DNA modification information. We analyzed the nanopore demo data in the adaptive sampling sequencing targeting the CpG islands and applied the linkage disequilibrium (LD) R² to identified methylation haplotype blocks (MHBs). We found that the cancer genome exhibited significantly smaller MHBs, higher CpG density, and a lower methylation LD R² value compared to normal cells. Additionally, we demonstrated the superiority of long-read sequencing in capturing large MHBs compared with short-read sequencing. By profiling the methylation changes near the JASPAR motif and actual chromatin immunoprecipitation sequencing (ChIP-seq) peaks, we also studied the epigenetic changes related to protein binding. Based on adaptive sampling technology, we conducted nanopore sequencing targeting regions with methylation quantitative trait loci (mQTLs) and genome-wide association study (GWAS) risk variants in the 22Rv1 cell line. After analyses, we inspected the closest haplotype-specific methylated region near the variant and identified allele-specific methylated regions with allele-specific accessibility signals in the ATAC-seq data. This study demonstrates the feasibility of nanopore sequencing for methylome profiling while preserving haplotype information, offering an innovative approach to elucidate the epigenetic changes driven by noncoding variants in the human genome.