Complete reference genome and pangenome improve genome-wide detection and interpretation of DNA methylation using...

Area of Science:

• Genomics and Epigenetics
• Human Genome Assembly
• DNA Methylation Analysis

Background:

• Existing human genome references (e.g., GRCh38) have limitations in comprehensively capturing genetic diversity and CpG sites.
• DNA methylation (DNAm) studies are crucial for understanding gene regulation and disease, but are constrained by reference genome accuracy.
• The development of the complete telomere-to-telomere human genome assembly (T2T-CHM13) and the human pangenome offers potential improvements.

Purpose of the Study:

• To evaluate the impact of T2T-CHM13 and the human pangenome reference on DNA methylation profiling.
• To assess improvements in CpG calling, probe accuracy, and the identification of unambiguous probes for DNAm arrays.
• To demonstrate the utility of these new references in epigenome-wide association studies (EWASs), particularly in cancer research.

Main Methods:

• Applied four short-read DNAm profiling methods to T2T-CHM13 and GRCh38 for comparative CpG calling.
• Evaluated probe cross-reactivity and mismatch using Illumina DNAm arrays with the new references.
• Utilized the human pangenome reference to expand CpG calling in short-read sequencing data and identify population-specific probes.

Main Results:

• T2T-CHM13 identified 7.4% more CpGs genome-wide compared to GRCh38 across tested DNAm profiling methods.
• Improved evaluation of probe cross-reactivity and mismatch, leading to more reproducible sets of unambiguous probes.
• The pangenome reference further increased CpG calling by 4.5% and identified population-specific unambiguous probes.

Conclusions:

• T2T-CHM13 and the human pangenome reference significantly enhance the accuracy and scope of DNA methylation studies.
• These advanced references facilitate the discovery of novel, biologically relevant DNAm alterations in EWASs.
• The findings support the practical application of T2T-CHM13 and pangenome for advancing genome biology and epigenetics research.