What are telomeres and how important are they for?

Telomeres consist of 5-15kb repetitive DNA sequences (TTAGGG in humans) that serve as protective caps, shielding chromosomes from damage and maintaining genomic stability. However, telomeres primarily rely on the enzyme telomerase for synthesis, and their length is not constant. In somatic cells, except for germ cells and stem cells, the lack of telomerase activity leads to gradual telomere shortening (approximately 50-200 bp) with each cell division. Studies have shown that telomeres in peripheral blood mononuclear cells (PBMCs) exhibit age-related shortening of 1190 bp per year during early life, 126 bp per year during childhood, and 43 bp per year during adulthood. As individuals age, telomere shortening slows down from early life to adulthood. When telomeres become very short, functional impairment occurs, triggering DNA damage response, cellular senescence, and a range of diseases. Therefore, the gradual shortening of telomere length in normal human somatic cells has emerged as a promising biomarker for age-related diseases.

The limitations of current methods and the innovation of long-read sequencing

However, current telomere measurement techniques still cannot achieve high-throughput detection of telomeres at single-base resolution or accurately quantify telomere length. Such as Terminal Restriction Fragment (TRF) analysis, real-time quantitative polymerase chain reaction (qPCR), High-Throughput Single Telomere Length Analysis (HT-STELA), and High-Throughput Quantitative Fluorescence in Situ Hybridization (HT-QFISH) can only provide average or relative lengths of telomeres, lacking single-base resolution. Most importantly, they lack the ability to detect telomere variant sequences (TVS) that may have profound implications for human aging and diseases. Therefore, there is an urgent need for a highly sensitive method capable of timely and longitudinal detection of individual telomere attrition in both experimental and clinical research.

Long-read sequencing platforms enable direct sequencing of telomeric genomic DNA fragments, allowing for the absolute measurement of telomere length at individual chromosome ends. It offers significant advantages in telomere research:

1. 1. Single-Base Resolution: Long-read sequencing enables direct sequencing of telomeric genomic DNA fragments, allowing for the precise measurement of telomere length at individual chromosome ends with single-base resolution. This provides a more accurate and detailed understanding of telomere dynamics compared to traditional methods.
2. 2. Detection of Telomere Variant Sequences (TVS): Long-read sequencing can identify and characterize telomere variant sequences, which are variations or mutations within the telomeric DNA sequence. Detecting these variants is crucial as they can have important implications for human aging and diseases.
3. 3. Highly Sensitive and Timely Detection: Long-read sequencing techniques are highly sensitive and can capture even subtle changes in telomere length. This allows for timely detection and longitudinal monitoring of individual telomere attrition in both research and clinical settings.
4. 4. Complete Telomere Profiling: Long-read sequencing provides a comprehensive view of telomere length across all chromosome ends. This enables researchers to examine telomere dynamics throughout the genome, identify variations in telomere length between chromosomes and regions, and gain a deeper understanding of telomere biology.

How can long-read sequencing be used for telomere research?

A study titled "High-throughput telomere length measurement at nucleotide resolution using the PacBio high fidelity sequencing platform", published in Nature Communication, showcases the cutting-edge capabilities of this technology. Researchers leveraged the PacBio platform’s ability to sequence long DNA fragments, culminating in precise measurements of telomere length, down to the level of individual nucleotides. This approach offers unprecedented accuracy and granularity in studying telomere dynamics.

1. 1. PacBio HiFi sequencing demonstrates high accuracy in sequencing telomeric repeat DNA. To validate the reliability of PacBio HiFi sequencing for measuring telomere length, researchers first performed HiFi sequencing on linearized DNA vectors (pWY82) containing Arabidopsis telomeric repeats (5’TTTAGGG-3′). They obtained highly accurate sequencing data for the vectors, with a median accuracy of over Q40 (99.99%) for CCS reads and an average of Q29 (99.87%). Alignment analysis using HiFi reads with accuracy above Q20 showed close to 100% alignment accuracy in the non-repeat regions and >95% in the repeat regions. Sequencing errors at the single-base level in the telomeric repeat sequence were similar to those in the non-repeat regions of the vectors. Overall, sequencing accuracy was higher in the non-repeat sequence regions, validating PacBio HiFi sequencing as a reliable method for detecting telomeric DNA fragments.
2. 2. High-throughput telomere length measurement using PacBio HiFi sequencing is reliable. Telomere length in peripheral blood leukocytes (PBL) or peripheral blood mononuclear cells (PBMC) is often used as a biomarker in aging-related research. To enable high-throughput telomere length measurement using PacBio HiFi sequencing, researchers performed mixed-sample sequencing of PBL samples from 104 patients in two runs of the HiFi sequencing platform. Each patient sample yielded a minimum of 2000 HiFi reads after splitting. Subsequently, the researchers calculated the average telomere length or original telomere length for each patient and examined its correlation with age. Consistent with expectations, telomere length or original telomere length showed a negative correlation with increasing age. These results indicate that PacBio HiFi sequencing is a reliable method for evaluating telomere length at the single-base resolution level.

In conclusion, long-read sequencing platforms offer valuable capabilities in telomere research, providing single-base resolution, detecting telomere variant sequences, enabling sensitive and timely detection, facilitating complete telomere profiling, and allowing integration with other genomic analyses. These advancements contribute to a better understanding of telomere dynamics and their implications for human aging and diseases.

Reference:

1. Jacob, Naduparambil K., Rose Skopp, and Carolyn M. Price. "G-overhang dynamics at Tetrahymena telomeres." The EMBO journal 20.15 (2001): 4299-4308.
2. Tham, CY., Poon, L., Yan, T. et al. High-throughput telomere length measurement at nucleotide resolution using the PacBio high fidelity sequencing platform. Nat Commun 14, 281 (2023). https://doi.org/10.1038/s41467-023-35823-7