High resolution long-read telomere sequencing reveals dynamic mechanisms in aging and cancer

Abstract

Telomeres are the protective nucleoprotein structures at the end of linear eukaryotic chromosomes. Telomeres' repetitive nature and length have traditionally challenged the precise assessment of the composition and length of individual human telomeres. Here, we present Telo-seq to resolve bulk, chromosome arm-specific and allele-specific human telomere lengths using Oxford Nanopore Technologies' native long-read sequencing. Telo-seq resolves telomere shortening in five population doubling increments and reveals intrasample, chromosome arm-specific, allele-specific telomere length heterogeneity. Telo-seq can reliably discriminate between telomerase- and ALT-positive cancer cell lines. Thus, Telo-seq is a tool to study telomere biology during development, aging, and cancer at unprecedented resolution.

Fig. 1. Telo-seq measures bulk and chromosome arm-specific telomere length.

a Schematic overview of Telo-seq protocol. b Terminal restriction fragment (TRF) analysis of HG002. c Violin plot of HG002 Telo-seq telomere length measurements. Violin represents telomere length distribution in kilobases (kb). Boxplot shows the median telomere length with interquartile range (IQR) and whiskers represent 1.5-fold IQR. The number of telomeric reads per sample is shown above the plot. Statistical analysis two-sided t-test with Bonferroni correction was performed and the adjusted p value is shown. d HG002 Telo-seq telomere length distribution with the mean telomere length shown as the dotted line. e Boxplot of the chromosome arm-specific telomere lengths of both HG002 replicates. The middle line represents the median telomere length, box the IQR, and whiskers 1.5-fold IQR. For (c–e), the results of two independent HG002 replicates are shown. For limitations on Telo-seq chromosome arm-specific telomere length assignment, see “Discussion”. For more details, see Supplementary Table 1, Supplementary Data 1 and Supplementary Figs. 1, 2. TRF: one experiment; Telo-seq: two experiments. Source data are provided as a Source Data file.

Fig. 2. Telo-seq resolves telomere shortening.

a Terminal restriction fragment (TRF) analysis of human IMR90 ^E6E7 fibroblasts at different population doublings (PDs). b Violin plot of IMR90 ^E6E7 Telo-seq telomere length measurements in kilobases (kb) at indicated PDs. Violin represents telomere length distribution. Boxplot shows the median telomere length with interquartile range (IQR) and whiskers represent 1.5-fold IQR. The number of telomeric reads per sample is shown above the plot. Statistical analysis two-sided t-test with Bonferroni correction was performed and adjusted p values are shown. c IMR90 ^E6E7 Telo-seq telomere length distribution at different PDs with the mean telomere length shown as the dotted line. d Bar graph showing the percentage of binned telomere length in IMR90 ^E6E7 fibroblasts at different PDs. e Linear regression analysis of IMR90 ^E6E7 mean telomere length against PDs. The blue line represents the best fit and the black curves have 95% confidence intervals. f Boxplot of chromosome arm-specific IMR90 ^E6E7 telomere length at different PDs. The middle line represents the median telomere length, box the IQR, and whiskers 1.5-fold IQR. For limitations on Telo-seq chromosome arm-specific telomere length assignment, see “Discussion”. For more details, see Supplementary Table 2, Supplementary Data 2 and Supplementary Fig. 3. One experiment per sample. Source data are provided as a Source Data file.

Fig. 3. Allele-specific telomere length differs in HG002.

Boxplot of allele-specific telomere length in kilobase (kb) of HG002 based on mapping against the haplotyped HG002 reference genome. The middle line represents the median, box the interquartile range (IQR), and whiskers the 1.5-fold IQR with outliers shown as black points. Individual telomere reads are shown as blue (paternal) and red (maternal) points. The paternal alleles of chromosome 13p and 22p are missing due to not passing the mapping quality filter. For more details, see Supplementary Data 3, Supplementary Fig. 4a and Methods. Pooled data from two independent experiments. Source data are provided as a Source Data file.

Fig. 4. Telomeres shorten with age.

a Violin plots of donor-derived fibroblast Telo-seq telomere length measurements in kilobases (kb). Violin represents telomere length distribution. Boxplot shows the median telomere length with interquartile range (IQR) and whiskers represent 1.5-fold IQR. The number of telomeric reads per sample is shown above the plot. b Bar graph showing the percentage of binned telomere length in donor-derived fibroblasts. c Linear regression analysis of fibroblast mean telomere length against donor age. The blue line represents the best fit and the black curves have 95% confidence intervals. d Violin plots of donor-derived fibroblast and matched induced pluripotent stem cells (iPSC) Telo-seq telomere length measurements. Violin represents telomere length distribution. Boxplot shows the median telomere length with IQR and whiskers represent 1.5-fold IQR. The number of telomeric reads per sample is shown above the plot. Statistical analysis two-sided t-test with Bonferroni correction was performed and adjusted p values are shown. e Bar graph showing the percentage of telomeres longer than 10 kb of donor-derived fibroblasts and their matched iPSC. For more details, see Supplementary Tables 3, 4, Supplementary Data 4, 5 and Supplementary Figs. 5,6. One experiment per sample. Source data are provided as a Source Data file.

Fig. 5. Telo-seq resolves TERT⁺ and ALT⁺ cancer cell telomere length distributions.

a Terminal restriction fragment (TRF) analysis of the indicated cancer cell lines. b Violin plot of cancer cell line Telo-seq telomere length measurements. Telomeres shorter than 80 kb are shown. Violin represents telomere length distribution. Boxplot shows the median telomere length with interquartile range (IQR) and whiskers represent 1.5-fold IQR. The number of telomeric reads per sample is shown above the plot. c Density plot of telomere length distributions measured by Telo-seq in indicated cancer cell lines. Telomere length in base pairs (bp) is log10 transformed. d Scatter plot of coefficient of variations to mean telomere length in kb of indicated cancer cell lines. For more details, see Supplementary Tables 5, 6. TRF: one experiment; Telo-seq: one, two, or four experiments. See Supplementary Table 6 for more information. Source data are provided as a Source Data file.