High-throughput telomere sequencing using PacBio HiFi sequencing

Telomeres are specialized nucleoprotein structures at the ends of linear chromosomes that are essential for the stability as well as the complete replication of the human genome. The progressive shortening of steady state telomere length in normal human somatic cells is a promising biomarker for age-associated diseases. However, there remain substantial challenges in quantifying telomere lengths in clinics as well as research laboratories due to the lack of accurate and high-throughput telomere length measurement method. Here, we describe newly designed telobaits that are complementary to the telomeric single-stranded G-rich overhangs, and can specifically and efficiently capture full-length telomeres for single-molecule real-time (SMRT) sequencing. We show that the telomere length measurement can be achieved at nucleotide resolution using the PacBio high fidelity (HiFi) sequencing platform. This new method provides the necessary accuracy and throughput to uncover the steady state telomere length distribution in human culture cell lines as well as clinic patient samples. Our results also reveal the extreme heterogeneity of telomeric variant sequences (TVSs) that dispersed throughout the telomere repeat region. Importantly, the unique distribution pattern and sequence of TVSs in individuals may be used as a fingerprint for sample identification. In addition, the presence of TVSs disrupts the continuity of the canonical (5'-TTAGGG-3')n telomere repeats, which could affect the binding of shelterin complex at the chromosomal ends. Therefore, TVSs may act as genetic polymorphisms that impair telomere protection; together with the absolute telomere length in each individual, TVSs may have profound implications for human aging and diseases. Our method could be a valuable tool not only in population-wide epidemiology studies but also for the longitudinal assessment of telomere attrition in individuals.