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.

Transposable elements (TEs) constitute nearly half of the human genome and shape chromatin organization, gene regulation and genome evolution. We have large gaps in understanding their influence to physiology and pathology for now. In humans, the most active elements—LINE-1 (L1), Alu, and SVA retain some copies with the ability to evade epigenetic repression and mobilize via target-primed reverse transcription (TPRT), whereas copies become inactive through fragmentation, mutation, or nesting, a process where a TE segments integrates into another TE segment. TE activity contributes to genomic instability and has been implicated in aging, cancer, neurological disorders, chromatin organization, and epigenetic regulation. Studying TEs is challenging due to their repetitive and polymorphic nature. Recent advances in sequencing technologies, including short- and long-read platforms, combined with specialized bioinformatic pipelines, now allow more comprehensive characterization of TE insertions, deletions, expression, and epigenetic status. Computational approaches vary in sensitivity, specificity, and resource requirements, and their performance is influenced by sequencing modality, coverage, and the reference genome used. Assembly-based and read-based methods, as well as tools integrating methylation or single-cell data, provide complementary insights into TE biology. Here we review the biology of active human TE. Survey state of the art short and long-read pipelines for TE analysis. And highlight their applications in studies of aging cancer and other complex diseases. We also provide practical guidance for selecting appropriate sequencing strategies and tools for TE-focused projects, and discuss emerging approaches and open questions in the field.

Transposable elements (TEs) account for nearly half of the human genome and shape chromatin organization, gene regulation, and genome evolution. However, their contributions to human physiology and disease remain incompletely understood. The most active elements in humans, LINE-1 (L1), Alu, and SVA, retain some copies with the ability to evade epigenetic repression and mobilize via target-primed reverse transcription (TPRT), whereas copies become inactive through various fragmentations and mutations. TE activity contributes to genomic instability and has been implicated in aging, cancer, neurological disorders, chromatin organization, and epigenetic regulation. Studying TE is challenging due to their repetitive and polymorphic nature. Recent advances in sequencing technologies and short- and long-read sequencing platforms, combined with specialized bioinformatic pipelines, currently enable more comprehensive characterization of TE insertions, deletions, expression, and epigenetic status. Computational approaches vary in sensitivity, specificity, and resource requirements, and their performance is influenced by sequencing modality, coverage, and the reference genome used. Assembly-based and read-based methods, as well as integrating methylation data or single-cell data, provide complementary insights into TE biology. This review summarizes the biology of active human TE, surveys state-of-the-art short- and long-read pipelines for TE analysis, and highlights their applications in studies of aging, cancer, and other complex diseases. We also provide practical guidance for selecting appropriate sequencing strategies and tools for TE-focused projects, and discuss emerging approaches and open questions in the field.