Abstract 1420: Long-read sequencing of single-stranded DNA of colorectal cancer patients

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Abstract Introduction: The presence of single-stranded (ss) DNA in biological samples has been known for many years, but whole-genome level ssDNA sequencing has only recently become feasible due to advancements in technology. Aims & Methods: To investigate the potential role of ssDNA further, we measured the ss/dsDNA ratio and fragment length distribution of nearly 200 human biopsy, normal adjacent tissue (NAT) and peripheral buffy coat (BC) samples. This included samples from healthy patients (n=31), as well as individuals suffering from inflammatory bowel disease (n=13), colorectal adenoma (n=53) and carcinoma (n=97).A small subset of these were subjected to a dual enrichment method: negative selection via dsDNA digestion is followed by positive selection by AMPure XP beads to yield gDNA enriched in the ss form, which was polyA-tailed and reverse transcribed into an ssDNA-cDNA hybrid, that we subjected to library preparation and whole-genome sequencing on Oxford Nanopore’s PromethION platform using various modalities: copy number variations, structural variants, and methylation profiling (5mC and 5hmC). We compared the ds- and ssDNA results of matched samples to highlight key differences between the two forms. Results: ssDNA concentration was 214 (±14.8) ng/ul, with an integrity (DIN) of 4.45 (±0.13) and length of 14.53 (±1.53) kbs. In the case of dsDNA, this was 152 (±14.6) ng/ul, 7.84 (±0.9) and 59.11 (±3.7) kbps, respectively. Average ss/dsDNA concentration ratio was 2.5 (±0.18), with a few samples having equal amounts, while most of them have at least twice as much single-stranded form. After sequencing, the average coverage was 20x (± 2.14) for dsDNA and 7.33 (±1, 17) for ssDNA, with a read length of 4.5 (±0.26) and 2.01 (±0.16) kilobases, consistent with the more fragmented condition of the ss form. CpG 5mC methylation of ssDNA is 43.4% (±2.6) of dsDNA (86.9% when considering one strand is synthetic), which is significant hypomethylation (p<0.05). 5hmC methylation ratio is doubling from 4.0% (±1%) on dsDNA to 9.5% (±1%) on ssDNA samples (p=0.006). However, we can only detect 1.5% of transversions and transitions, and 0.5% of indels when normalized for coverage differences - strongly uneven representation of the genome in the ssDNA fraction might explain this finding. Conclusion: Third generation long-read sequencing is a promising and flexible method that can be adapted to examine the ssDNA fraction of the genetic material, providing potential new insights into the background of pathologies. Citation Format: Bela Molnar, Kristóf Rada, Nikolett Szakállas, Barbarad K. Barták, Alexandra Kalmár, István Takács. Long-read sequencing of single-stranded DNA of 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 1420.

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