Abstract 4104: Adapter-anchored methylation specific amplification on genomic DNA using proximity primers (aMAPP)

Purpose: The epigenetic deregulation of CpG islands (CGIs) plays a crucial role in cancer initiation and progression. Genome-wide analysis of aberrant methylation patterns in CGIs offers a promising approach for the early cancer detection. However, the clinical utility of whole genome bisulfite sequencing is limited by the need for high sequencing coverage to achieve sensitive detection. To address this challenge, we have developed aMAPP, a method that enriches hypermethylated and/or hypomethylated CGIs. This approach enables the detection and tracing of aberrant methylation patterns, achieving high coverage in CGIs with ultra-low depth sequencing. Methods: NEBNext Ultra II Library Prep. was used for adapter ligation. DNA was then subjected to APOBEC-mediated deamination and/or TET-Assisted Pyridine Borane Sequencing (TAPS) and pre-amplified. Methylation-specific amplification was then conducted using novel, customized proximity primers composed of an anchor, binding to the adapter, and a 6-10 bp probe, designed to bind tandem CpG dinucleotides downstream of the adapter. These primers amplify selectively DNA fragments containing methylated CpGs that remain intact after APOBEC deamination or unmethylated CpGs that remain intact after TAPS. Following Proximity Primer-PCR, MiSeq was conducted, generating approximately 500,000 reads per sample. Results: Proximity primers were shown to amplify APOBEC-treated DNA fragments containing CpGs that match the primer probes, even if these CpGs are 100 bases downstream the adapter. While unmethylated fragments lack CpGs and are not amplified (∆Ctmeth-unmeth≈18). Applying aMAPP to APOBEC-treated methylated gDNA provided large amplification footprint of genome-wide methylated CGIs with remarkable efficiency, yielding 355-fold and 746-fold more CGIs with coverage of ≥5 and ≥10, respectively, compared to standard EM-Seq. Depending on conditions applied, aMAPP captures >80% of all CGIs with high coverage using ultra-low sequencing depth. Similarly, aMAPP applied to TAPS-treated unmethylated DNA captured selectively the unmethylated CGIs. aMAPP can be applied with very low starting material (10-100pg). Upon application to clinical samples, aMAPP detected thousands of CGIs and identified CpG island methylator phenotype (CIMP) and the top 20 hypermethylated CGIs in colorectal cancer. aMAPP could trace differentially methylated CGIs (DMR) in tumor samples present at 0.01% levels within adjacent normal tissue, or within circulating DNA of normal volunteers. Conclusion: aMAPP enriches methylated and/or unmethylated portions of the genome with a unique efficiency, simplicity and flexibility that supersedes previously reported methylation enrichment approaches, enabling focused methylation sequencing at high coverage. Multiple applications in biomarker tracing and discovery can be envisioned in cancer, and beyond cancer. Citation Format: Farzaneh Darbeheshti, Hayet Radia Zeggar, Hamzeh Salmani, Yibin Liu, Viktor A. Adalsteinsson, G. Mike Makrigiorgos. Adapter-anchored methylation specific amplification on genomic DNA using proximity primers (aMAPP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4104.