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

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, through the 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 slow clonal variation or may represent a new biological mechanism that maintains equilibrium unique to each chromosome 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, tel1 Delta rif1 Delta double mutants had a very small, but significant, increase in telomere length compared with the tel1 Delta single mutant, suggesting an influence of Y ' elements on telomere length regulation. We sequenced telomeres in a telomerase-null mutant (est2 Delta) and found the minimal telomere length to be similar to 75 bp. In these est2 Delta mutants, there were apparent telomere recombination events at individual telomeres before the generation of survivors, and these events were significantly reduced in est2 Delta rad52 Delta double mutants. The rate of telomere shortening in the absence of telomerase was similar across all chromosome ends at similar to 5 bp per generation. This new method gives quantitative, high-resolution telomere length measurement at each individual chromosome end and suggests possible new biological mechanisms regulating telomere length.