Long-Read Sequencing Reveals Heterogeneous LINE-1 Insertion and Methylation Patterns in Colorectal Cancer

Background/Objectives: LINE-1 (L1) retrotransposons comprise approximately 17% of the human genome. Most are truncated, rearranged, or mutated molecular fossils, and only a small, variably estimated subset of L1 loci retains retrotransposition competence. L1 hypomethylation and retrotransposition have been associated with colorectal cancer (CRC), but comprehensive characterization of individual L1 insertions and their methylation remains challenging because of their repetitive sequence and length. In particular, conventional short-read sequencing provides limited resolution of individual L1 loci. Methods: We used Oxford Nanopore long-read whole-genome sequencing (WGS) to characterize L1 insertion-burden, insertion structure, and methylation in 21 CRC tissue samples, 22 normal adjacent tissue samples (NAT), 13 CRC-negative control tissues (NEG), and single samples of the CRC-derived cell lines SW480 and HT29. We focused on L1HS, the human-specific LINE-1 subfamily, and combined structural insertion detection with native DNA methylation profiling of individual full-length L1HS loci. Results: We identified 5,163 L1HS insertions meeting stringent predefined criteria, including 1,030 full-length insertions. L1HS insertion-burden showed a significant increase from NEG to NAT and CRC tissue groups (Jonckheere–Terpstra, P = 0.0338), while CRC tissues exhibited significantly lower mean L1HS methylation (Kruskal–Wallis \( P = 1.7 \times 10^{-5} \)) and substantial inter-sample and locus-level heterogeneity. Global mean L1HS methylation showed a weak but significant correlation with the number of full-length L1HS insertions, whereas the minimum methylation observed among L1HS loci within each sample showed a stronger correlation with L1HS insertion-burden. Conclusions: These findings demonstrate the utility of long-read sequencing for integrated structural and epigenetic characterization of individual L1HS loci in CRC and provide a framework for future investigation of LINE-1 features in larger, molecularly and clinically characterized cohorts.