Abstract 329: NanoTelo: A workflow for detecting telomere absolute length based on nanopore sequencing technology

Abstract Background: Telomeres are specialized nucleoprotein structures located at the end of linear chromosomes in eukaryotic cells, and protect the ends of chromosomal DNA from degradation, end-to-end fusion and abnormal recombination. Changes in telomere length have become an important biomarker, which is closely related to cellular aging, all-cause mortality and a variety of diseases. However, there are still challenges in quantifying telomere absolute length, due to repeats of nucleotide sequence (5’-TTAGGG-3’) n in single-stranded G-rich telomeric region, and lack of a method for nucleotide separation and enrichment of individual telomeres. Methods: We developed NanoTelo, a workflow for detecting telomere absolute length based on nanopore sequencing technology. For experimental steps, a self-designed telomere adapter was first connected to the C-rich strand at the end of the chromosome by utilizing the 3' end overhang. And the 5' end of the telomere adapter was complementary to the sequencing adapter, allowing the antisense telomere chain to perform single-strand extension sequencing in the 5' to 3' direction, thus into the sub-telomeric region from the telomere end. The sequencing library was nanopore-sequenced on the QPursue-6k according to the manufacturer’s instructions. For data analysis, reads were strictly extracted (dataset B) with the telomere adapter and library structure in raw sequencing data (dataset A). The reads of dataset A and dataset B were corrected, aligned to the human reference genome (T2T-CHM13v2. 0), and calculated the telomere length distribution of each chromosomal (autosomes and sex chromosomes) p/q-arm, respectively. Defined the mean telomere length of all chromosomes represented the overall telomere length, the estimated results of dataset A and dataset B were weighted to obtain the final telomere absolute length. Results: We selected 5 cell lines, which was HCT116, 293T, T24, IMR90 and WI38, and performed 2 experimental replicates for each one. We found that the estimated telomere lengths for each cell line were 3, 528/3, 512 bp, 4, 298/4, 311 bp, 2, 745/2, 685 bp, 4, 926/4, 915 bp and 4, 143/4, 096 bp, respectively. A comparative analysis with results from the Pacbio sequencing platform for each cell line in the Tham et al. 's paper, showed that there was no significant statistical difference (two-tailed T-test: p-value=0. 98), and the mean absolute difference for estimated telomere length between the two platforms was 7. 7 bp (95% CI: 5. 4-10. 0 bp). Conclusions: NanoTelo demonstrated the possibility of measuring telomere absolute length with a single chromosomal p/q-arm at nucleotide-level resolution. We hope that NanoTelo can not only be used as an important tool for estimating telomere length to dynamically monitor aging, but also provide strong technical support for scientific research and/or clinical application related to human anti-aging and disease intervention. Citation Format: Jidong Lang, Yanmei Chen, Zhi Yang, Beibei Huo. NanoTelo: A workflow for detecting telomere absolute length based on nanopore sequencing technology abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 329.