Telomeres and Aging: Insights from Long-Read Sequencing

At a glance:

• What are telomeres?
• Limitations of traditional telomere measurement methods
• Research progress and new technology development
• Conclusion

Located at chromosome termini within eukaryotes, telomeric regions play crucial roles in preserving chromosomal integrity and regulating cellular replication. In this analysis, we examine a pair of innovative studies that leverage advanced sequencing platforms - specifically Oxford Nanopore and PacBio HiFi technologies - to establish novel approaches for telomere analysis. These methodologies provide unprecedented insights into telomere dynamics and their underlying molecular mechanisms, particularly in the context of aging-related processes, oncological developments, and various biological phenomena.

What are telomeres?

At the extremities of eukaryotic linear chromosomes lie specialized regions known as telomeres, which play crucial roles in maintaining chromosomal integrity and regulating cellular replication. These structures serve as biological timekeepers, with their dimensions indicating both past replication events and future proliferative capacity - functioning essentially as cellular life-span indicators. The progressive reduction in telomere size correlates with advancing age, subsequently triggering cellular senescence, programmed cell death, and potentially malignant transitions. The impact of telomeric dimensions on human well-being is profound, as both insufficient and excessive lengths can trigger pathological conditions. Research demonstrates a correlation between diminishing telomeres and both heightened disease susceptibility and decreased longevity. However, significant questions persist regarding the precise temporal dynamics and mechanisms of telomere degradation, including whether this process affects certain chromosomes disproportionately.

Figure 1. Telomere repeats at individual chromosome ends.(Aubert, G., et.al,2008)

Limitations of traditional telomere measurement methods

Scientists have employed numerous analytical techniques for assessing telomeric dimensions, encompassing methodologies such as TRF analysis, STELA, TeSLA, qPCR-based measurements, Q-FISH, Flow FISH, and computational approaches utilizing next-generation sequencing data. Nevertheless, inherent challenges posed by telomeric repetitiveness and extensive length have historically impeded precise characterization. Traditional approaches fail to effectively distinguish between specific chromosomal alleles or individual telomeric regions, resulting in constrained precision and reliability.

The landscape of telomere research has been transformed by recent technological breakthroughs in long-read sequencing platforms. These innovations now enable comprehensive examination of complete telomeric structures, including both their dimensional attributes and molecular composition. Advanced platforms, particularly Oxford Nanopore and PacBio HiFi technologies, have revolutionized the field by facilitating enhanced resolution in telomere sequencing and measurement capabilities.

Research progress and new technology development

Two research teams led by the Salk Institute for Biology and Stanford University in the United States used ONT's original growth read length sequencing technology to develop a breakthrough new technology, Telo-seq, which can solve the problem of telomere sequence analysis. Research telomere biology in development, aging and cancer with unprecedented resolution. In addition, the research team has developed a method for high-resolution, high-throughput measurement of individual intact telomeres using nanopore sequencing, which can promote understanding of telomere maintenance mechanisms and use of telomere length as a clinical biomarker for aging and disease.

Telo-seq technology

In an article titled "High resolution long-read telomeres sequencing reveals dynamic mechanisms in aging and cancer", the research team at the Salk Institute for Biological Research pioneered a new method-Telo-seq, which uses nanopore sequencing to effectively sequence human telomeres. It can determine the complete sequence and precise length of telomeres on a single chromosome, providing new insights into the dynamic changes of telomeres in health and disease. The research team used Telo-seq to analyze global, arm-specific and allele-specific telomere lengths and explored telomere shortening in five population doublings (PD).

The results showed that telomere lengths were heterogeneous within the same sample, and there were large differences in shortening rates; despite the heterogeneity, the telomere lengths of some chromosome arms were always long or short relative to the average telomere length, and these chromosome arm-specific telomere length differences were conservative among different individuals; compared with the elderly, telomere lengths in young people were usually longer, indicating that telomere lengths shorten with age. In summary, Telo-seq is an efficient and repeatable method that can reveal heterogeneity in telomere lengths within samples, chromosome arm-specific, and allele-specific.

Figure 2. Telo-seq measures the length of specific telomeres in chromosome arms.(Schmidt, T.T , et.al,2024)

In addition, the research team's analysis of cancer cells showed that Telo-seq can effectively distinguish between telomerase positive (TERT+) and telomere selective elongation (ALT+) positive cancer cell lines; it can analyze both short telomeres in normal replication-crisis cells, and ultra-long telomeres in ALT+ cancer cell lines. In the sense that ALT-positive cancers are often more aggressive and require a different treatment than telomera-positive cancers; in this sense, Telo-seq can be used as a rapid and reliable diagnostic tool to identify cancer types and guide more personalized treatment plans. At the same time, combined with whole-genome sequencing, Telo-se will also help study inherited telomere syndrome and address the impact of potential genetic changes on telomere structure.

Professor Jan Karlseder, corresponding author of the article, said: "The biggest impact of Telo-seq will be that it will open a new era of telomere research, which will answer questions about development, aging, stem cells and cancer that we cannot use previous tools to solve. I think what we are starting to understand now is actually just the tip of the iceberg, and as we learn more, Telo-seq technology will reveal more unknown biological mysteries.

Telometer technology

In another article titled "Digital telomere measurement by long-read sequencing distinguishes healthy aging from disease", the Stanford University research team developed a sequencing preparation and bioinformatics analysis process-Telometer, which can repeatedly measure telomeres from the whole genome or telomeres enriched long-read sequencing data, that is, digital telomere measurement (DTM) using nanopore sequencing to understand how the distribution of human telomere lengths changes with age and disease.

By aligning telomeric repeats with the chromosomal ends of the recently completed T2T human reference genome, the research team was able to identify reads containing telomeres; the range of each telomere is defined as the distance from the terminal repeats at the end of the chromosome to the last two consecutive repeats in front of the subtelomere sequence, which anchors the telomere region to its reference chromosome. It was verified that the average telomere length measured by long-read-length sequencing was highly correlated with the existing gold standard, TRF and Flow-FISH.

Figure 3. Nanopore long read sequencing is used for high resolution telomere measurement. (Sanchez, S.E , et.al,2024)

At the same time, as additional telomere measurements increase, the standard error of this method's measurement decays exponentially, eventually reaching a maximum accuracy of 30-40 base pairs. Subsequently, the research team measured telomere wear and neonatal elongation at a resolution of up to 30 bp in genetically defined human cell populations, blood cells from healthy donors, and blood cells from patients with genetic defects in telomere maintenance. The results show that human aging is accompanied by the gradual loss of long telomeres and the accumulation of short telomeres, while in patients with telomere maintenance defects, the accumulation of short telomeres is more pronounced and is associated with phenotypic severity.

In addition, based on the generated high-resolution measurement data set, the research team applied machine learning to train a binary classification model that can distinguish healthy individuals from telomere biological disorders (TBD) individuals; it is worth noting that DTM can distinguish asymptomatic carriers from normal individuals with high sensitivity and is expected to serve as a diagnostic or prognostic tool for assessing TBD.

Figure 4. Differentiating aging and disease in healthy people based on telomere length distribution. (Sanchez, S.E , et.al,2024)

To sum up, the research team used the DTM method to measure with high precision the telomere shortening observed in culture and in vivo for three TBD genotypes; and found that telomerase preferentially acts on the shortest telomeres in humans. As long-read genomic databases grow, analyzing telomere length distributions from larger, more diverse and ideal longitudinal studies will make telomere length a more powerful and possibly even a predictive biomarker of aging and disease.

Conclusion

The advancement of long-read sequencing technologies, such as Oxford Nanopore and PacBio HiFi, has revolutionized telomere research by enabling high-resolution, chromosome-specific telomere analysis. The development of Telo-seq and Telometer represents significant breakthroughs in understanding telomere dynamics in aging and disease. Telo-seq provides precise telomere length measurements at the single-chromosome level, uncovering heterogeneity in telomere shortening and distinguishing between different cancer subtypes. Meanwhile, Telometer leverages long-read sequencing for digital telomere measurement, offering a powerful diagnostic and prognostic tool for telomere-related disorders. These advancements not only deepen our knowledge of telomere biology but also pave the way for future applications in aging research, cancer diagnostics, and personalized medicine. As sequencing technologies continue to evolve, telomere length measurement may become an essential biomarker for health monitoring and disease risk assessment.