NCM 2023 Houston: Telomere dynamics in aging and cancer by nanopore long-read sequencing

Telomeres are the protective, nucleoprotein structure at the ends of linear eukaryotic chromosomes. The accurate measurement of both telomeric length and composition of individual telomeres in mammalian cells has been challenged by the length and repetitive nature of telomeres. With nanopore sequencing, it is now technically possible to sequence entire telomeres and map them to individual chromosome arms. Here, we report a reliable method to enrich, sequence and analyze human telomeres using nanopore sequencing. To enrich for telomeric sequences, we combine the ligation of adapters complementary to the telomeric G-overhang with restriction enzyme digestion to sequence the telomeric C-strand and part of the adjacent subtelomere. The subtelomeric information is harvested to map individual telomeric reads to specific chromosome arms. We have measured bulk and chromosome-arm specific telomere length dynamics during cellular aging of cultured primary cells and in a patient-derived aging cohort. To address the impact of the telomere maintenance mechanism on telomere length and composition, we have sequenced five well-established telomerase- and ALT-positive cancer cell lines. Our results suggest that, based on nanopore telomere long-read sequencing, ALT-positive cells can be easily discriminated from normal and telomerase-positive cancer cells. In summary, nanopore sequencing of telomeres grants a deeper understanding of individual telomere composition through telomere length measurement and mapping to specific chromosome arms. As such, telomere sequencing using our nanopore-based enrichment method is a valuable tool to study telomere biology in aging and cancer.