Abstract 218: Long-read nanopore sequencing in pursuit of complete methylation profiling of Hungarian colorectal cancer patients
Authors: Kristóf R. Rada, Nikolett Szakállas, Barbara Barták, Alexandra Kalmár, István Takács, Béla Molnár
Journal: Cancer Research (2025)

Abstract Introduction: Variations of the epigenetic landscape are the hallmark of many pathologies, including cancer. We investigated the Hungarian colorectal cancer cohort via Oxford Nanopore’s long-read whole-genome sequencing (WGS) platform to provide for the first time a genome-spanning methylation report of the country. A prominent benefit of the method is native, and so, bias-free detection of modified or alternative nucleotides. Aims &amp; Methods: We selected 136 tissue biopsy, resection, normal adjacent tissue (NAT) and peripheral buffy coat (BC) samples from patients of Semmelweis University, to represent the range of pathological progression: healthy - adenoma (AD) - carcinoma (CRC). After thorough quality control by fragment length analysis (Tape Station), WGS was carried out on Nanopore’s PromethION platform in simplex basecalling mode. Bioinformatic analysis utilized the Nanopore software package, Dorado basecaller and our custom scripts. We focused on methylation and hydroxymethylation of cytosine residues as the most abundant form of epigenetic marks. Results: Coverage is 19.3x (±0.13), with an average read length and precision of 4.3 (±0.16) kb and 98% (±0.01), respectively; median PHRED is 16.03 (±1.18) and N50 is 7807 (±2262). Average GC ratio is 47% (±7%), island-mapped CpG sites show 69% (±5%) average methylation, of which 5hmC accounts for 5.68% (±1.88%), showing significant variations between groups: from 71% (healthy individuals), average methylation drops by 4.1% (AD, p=0.013), 8.8% (CRC, p=0.007), and 12.9% (resection, p&amp;lt;0.001) in solid tumors; by 0.1% (AD) and 2.5% (CRC) in buffy coats (not significant); and by 2.7% (CRC, p=0.01), and 4.6% (resection, p&amp;lt;0.001) in NAT samples. Solid samples always show significant hypomethylation compared to buffy coats. Contrasting this, 5hmC reveals a different pattern: it is relatively stable in solid samples and buffy coats, but rises by 47% (CRC, p&amp;lt;0.001) and 56% (resection, p&amp;lt;0.001) in normal adjacent tissues. When matching the tumor and healthy tissue of the same patient, we also see significant hypomethylation: 5mC decreases by 7% (±2.1%, p=0.005), and 5hmC by 31.4% (±3.9%, p&amp;lt;0.001) with very few exceptions. Conclusion: long-read WGS is a strong tool for studying methylation in solid and liquid samples of colorectal cancer patients, revealing marked differences between solid and liquid samples, and also between tumor and healthy tissue. We corroborated the general cytosine hypomethylation associated with cancer, while also observing new trends in hydroxymethylation. Citation Format: Kristóf R. Rada, Nikolett Szakállas, Barbara Barták, Alexandra Kalmár, István Takács. Long-read nanopore sequencing in pursuit of complete methylation profiling of Hungarian colorectal cancer patients [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 218.