New Telomere Measuring Method Addressing Limitations Of Current Options
By Deborah Borfitz
March 8, 2023 | A research collaboration led by led by Duke-NUS Medical School and National Heart Centre Singapore (NHCS) has set the stage for telomere measurement to one day be added to the list of clinical-grade methods for assessing biological aging. The focus is on rapid and precise measurement of individual telomeres—the caps at the ends of chromosomes known to protect against early aging and disease—using a technique that employs “telobaits” that latch onto the ends of telomeres in large pools of DNA fragments so they can be fished out with scissor-like enzymes and have their DNA signature read by high-throughput genetic sequencing.
So explains Angela Koh, associate professor and senior consultant with the department of cardiology at the NHCS and associate professor with the SingHealth Duke-NUS Cardiovascular Sciences Academic Clinical Program. The extent to which telomeres correlate with structural and functional deterioration at the organ level is a “complex paradigm because every organ is different, with each aging at its own speed or not aging at all in some individuals.”
But a better understanding of telomeres with more reproducible and precise methods is a step in the right direction, she adds. Work recently published in Nature Communications (DOI: 10.1038/s41467-023-35823-7) furthers the journey to answers.
The new approach is “capable of measuring allele-specific telomere length with nucleotide resolution,” explains Shang Li, associate professor with the Duke-NUS Cancer and Stem Cell Biology Program. Telobaits are first used “to enrich genomic DNA fragments with the telomere sequence, so we can multiplex multiple samples in one sequencing run to achieve high throughput and reduce the cost.
“Next,” Li continues, “enzymes are used to trim the genomic DNA fragments to a smaller size that will be optimal for Pacific Biosciences HiFi sequencing [roughly 10-25 kilobases]. These trimmed fragments then undergo PacBio library preparation, followed by sequencing to generate accurate reads of the genomic DNA containing telomere sequence.” Data acquired from the PacBio sequencer are analyzed using an in-house bioinformatics pipeline, called Telomap, to estimate telomere length.
“Classical methods that are used for clinical purposes [Terminal Restriction Fragment analysis, Flow-FISH (fluorescence in-situ hybridization), and PCR-based approaches] can only provide the mean bulk lengths of telomeres, which masks the weakest link that is the shortest telomere,” says Li. “In addition, none of them can provide individual telomere length.”
In terms of measuring telomere length at individual chromosomal ends or to characterize the length distribution of individual telomeres, the new telomere length measurement technique has the added advantage of being easy to use and producing high throughput estimates—up to 72 patient samples in a single run so far.
Newer techniques like single telomere absolute-length rapid assay, high-throughput single telomere length analysis, and high-throughput quantitative fluorescence in situ hybridization can provide individual telomere length, Li adds. “However, they lack nucleotide resolution, exhibit a wider margin of error, and, importantly, are unable to distinguish which allele the telomere belongs to.” They also cannot detect telomeric variant sequences (TVSs).
Most significantly, Li says, their novel method can achieve “very good accuracy with increased sequencing depth.” As pointed out in the paper, the standard error of mean is less than +/-20 base pairs (bp) at a sequencing depth of 15,000 reads.
“Such accuracy is necessary for longitudinal tracking of the telomere attrition rate in individuals within a short time frame,” says Li, which in peripheral blood is estimated to be around 30 to 50 bp per year. “This is particularly relevant to [diet or exercise-related] clinical studies... that aim to slow down telomere attrition and attain healthy longevity.”
The research team designed a total of six telobaits with the most-probable sequences (hexanucleotide repeat) to anneal to the single-stranded DNA “G-rich overhang,” which is found at the end of all human telomeres, explains Li. Each of the telobaits contains a signature barcode for identification and combination analysis.
“Annealed and tagged telomere-containing DNA are then purified from the genomic DNA fragment pool using capturing microbeads,” continues Li. “Finally, a restriction enzyme called EcoRI is used to cut and release the telomere-containing DNA fragments from the microbeads and send these for DNA sequencing via the PacBio system.”
As schematically illustrated in the published paper, each of the six telobaits contain a single telomere repeat with all six possible ends as well as a unique barcode, EcoRI restriction endonuclease recognition site, and biotin labeling. Steps are then taken to enrich the telomere repeat-containing genomic DNA fragments using these telobaits in PacBio high fidelity (HiFi) sequencing.
At present, as the research team has demonstrated, it is possible to map the telomeric reads to nine chromosomal ends, reports Li. “Our data indicated that the distribution and sequence of TVSs for every chromosomal end in each individual are different.” With larger sample sizes and in-depth mechanistic studies of the TVSs, “this could potentially be used as a form of biological identification and lineage tracing in the forensic and evolutionary sciences.”
Li notes one important question in aging is the heterogeneity of the aging process in individuals. “Our studies have shown that the TVS signature [distribution and sequence] differs for each chromosomal end in different individuals. Additionally, we demonstrated that TVSs disrupt the binding of telomere sequence-specific binding proteins TRF1 and TRF2 [sub-unit of the so-called ‘shelterin complex’].”
In other words, the exact sequence of telomeres and not just their length may impact the binding and protection of chromosomal ends by the shelterin complex. One significant observation, says Li, is “the presence of larger and more abundant TVSs near the sub-telomeric region.” It could be that the binding density is lower when telomeres become extremely short, leading to “precipitative deterioration of telomere protection later in life.”
One of the team’s top goals, Li says, is to see TVS used as “a predictive biomarker for human aging and aging-related disease at the individual level and for population-level studies.” The researchers are currently collaborating with SingHealth clinicians on several ongoing healthy longevity trials aiming to validate their longitudinal telomere length measurement method to monitor aging in individuals.
The long-term goal, agree Li and Koh, is to develop the next clinical standard for telomere length measurement. They are also looking for commercial partners to speed up this process.
In addition to PacBio HiFi sequencing, the Oxford Nanopore platform could potentially be used with the novel telomere length-measurement application—at least once it can achieve accurate reads of each nucleotide for telomere profiling, says Li. “We are testing Nanopore at this moment.”