Work

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

Year: 2025

Type: article

Abstract: 5-methylcytosine (5mC) is the most common chemical modification occurring on the CpG sites across the human genome. Bisulfite conversion combined with short-read whole genome sequencing can capture and quantify the modification at single nucleotide resolution. However, the PCR amplification process can lead to duplicative methylation patterns and introduce 5mC detection bias. Additionally, the limited read length also restricts co-methylation analysis between distant CpG sites. The bisulfite conversion process presents a significant challenge for detecting variant-specific methylation due to the destruction of allele information in the sequencing reads. To address these issues, we sought to characterize human methylation profiling with nanopore long-read sequencing, aiming to demonstrate its potential for long-range co-methylation analysis with native modification call and intact allele information. In this regard, we first analyzed the nanopore demo data in the adaptive sampling sequencing run targeting all human CpG islands. We applied the linkage disequilibrium R2 to calculate the co-methylation in nanopore data, and further identified 27,875, 50,481, 26,542 and 51,189 methylation haplotype blocks (MHB) in COLO829, COLO829BL, HCC1395 and HCC1395BL cell lines, respectively. Although the majority of the co-methylation was in a short range (≤200bp), a small portion (1~3%) showed long distance (≥1,000bp), suggesting potential remote regulatory mechanisms across the genome. To characterize the epigenetic changes related to transcription factor binding, we profiled the 5mC percentage changes surrounding various motif sites in the JASPAR collection. This analysis showed reduced methylation at CTCF, NRF1 and ZNF93 binding sites but increased methylation at RARA sites. To further investigate allele-specific 5mCpG in the prostate genome, we performed an adaptive nanopore sequencing by targeting genomic regions covering methylation quantitative trait loci (mQTL) and genome-wide association study (GWAS) risk germline variants in the 22Rv1 cell line. After long-read based phasing, we compared the 5mCpG signals between the two haplotypes and identified 6,390 haplotype-specific methylated regions in the cell line (p-MWU ≤ 1 × 10^-5 and delta ≥ 50%). By examining haplotype-specific methylated regions near the phasing variants, we identified examples of allele-specific methylated regions that showed allele-specific accessibility in the ATAC-seq data. The methylation levels were negatively correlated with chromatin accessibility at the genome-wide scale. Our study demonstrates the feasibility of nanopore sequencing for native methylome profiling while preserving haplotype information, offering a novel approach to uncover the regulatory mechanisms of the human genome.

Source: Human Genetics and Genomics Advances

DOI: 10.1016/j.xhgg.2025.100532

Authors: Yijun Tian, Shannon K. McDonnell, Lang Wu, Nicholas B. Larson, Liang Wang

Institutions: Moffitt Cancer Center, Mayo Clinic, University of Hawaiʻi at Mānoa, University of Hawaii Cancer Center, Louisiana State University Health Sciences Center New Orleans

Language: English

Topic: Epigenetics and DNA Methylation

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,300

Funders: National Institutes of Health, National Cancer Institute