Chromosome specific telomere lengths and the minimal functional telomere revealed by nanopore sequencing

Abstract

We developed a method to tag telomeres and measure telomere length by nanopore sequencing in the yeast S. cerevisiae. Nanopore allows long read sequencing through the telomere, subtelomere and into unique chromosomal sequence, enabling assignment of telomere length to a specific chromosome end. We observed chromosome end specific telomere lengths that were stable over 120 cell divisions. These stable chromosome specific telomere lengths may be explained by stochastic clonal variation or may represent a new biological mechanism that maintains equilibrium unique to each chromosomes end. We examined the role of RIF1 and TEL1 in telomere length regulation and found that TEL1 is epistatic to RIF1 at most telomeres, consistent with the literature. However, at telomeres that lack subtelomeric Y' sequences, tel1Δ rif1Δ double mutants had a very small, but significant, increase in telomere length compared to the tel1Δ single mutant, suggesting an influence of Y' elements on telomere length regulation. We sequenced telomeres in a telomerase-null mutant (est2Δ) and found the minimal telomere length to be around 75bp. In these est2Δ mutants there were many apparent telomere recombination events at individual telomeres before the generation of survivors, and these events were significantly reduced in est2Δ rad52Δ double mutants. The rate of telomere shortening in the absence of telomerase was similar across all chromosome ends at about 5 bp per generation. This new method gives quantitative, high resolution telomere length measurement at each individual chromosome end, suggests possible new biological mechanisms regulating telomere length, and provides capability to test new hypotheses.

142 telomere-to-telomere assemblies reveal the genome structural landscape inSaccharomyces cerevisiae

“…14). The two same chromosome extremities were also described as the longest in W303, (Sholes et al ., 2021). The conservation of larger telomere size at TEL03L at the population level suggests that the underlying genetic determinants would be conserved since the species diverged from its last common ancestor at least ~180 KYA (see above).…”

“…This model implies that all individual telomeres would gradually converge toward the population mean length distribution after a sufficiently large number of divisions and that the overall telomere length distribution would converge to a steady state (Bianchi and Shore, 2008; Xu et al ., 2013). However, a recent study that used nanopore sequencing to measure individual telomere length reported that in W303, each chromosome end could have a specific and reproducible telomere length distribution that would remain stable over at least 120 generations (Sholes et al ., 2021). We examined telomere length variation between individual chromosome ends across the population of 100 strains.…”

High-throughput telomere length measurement at nucleotide resolution using the PacBio high fidelity sequencing platform

“…Given the slow and progressive shortening of telomeres in adulthood, a highly sensitive method that can longitudinally detect telomere attrition in an individual in time is needed for both experimental and clinical studies 34 . In this regard, the recent development of long-read sequencing platforms has enabled the direct sequencing of telomere-containing genomic DNA fragments to obtain absolute telomere length at individual chromosome end [35][36][37][38][39] . However, current methods require the sequencing of whole genome to analyze the individual's telomere distribution, which precludes high-throughput capability.…”

Effects of DNA extraction, DNA integrity, and laboratory on the precision of qPCR-based telomere length measurement - a multi-lab impartial study

“…There is a clear need to better understand how TL measurement, using any assay, is impacted by polymorphic variation in subtelomeric regions and interstitial telomeric repeats. The use of direct sequencing via nanopore technology offers the unique possibility of generating a true "gold" standard TL measurement (25). Further, the completion of the sequencing of the human genome, telomere to telomere, is an opportunity to define the unique DNA sequences bridging the subtelomeric region and the telomere for each chromosome arm and leverage existing technologies such as STELA to determine the specific length of each unique chromosome (26).…”