Human telomere length is chromosome end-specific and conserved across individuals

Abstract

Short telomeres cause age-related disease, and long telomeres contribute to cancer; however, the mechanisms regulating telomere length are unclear. We developed a nanopore-based method, which we call Telomere Profiling, to determine telomere length at nearly single-nucleotide resolution. Mapping telomere reads to chromosome ends showed chromosome end-specific length distributions that could differ by more than six kilobases. Examination of telomere lengths in 147 individuals revealed that certain chromosome ends were consistently longer or shorter. The same rank order was found in newborn cord blood, suggesting that telomere length is determined at birth and that chromosome end-specific telomere length differences are maintained as telomeres shorten with age. Telomere Profiling makes precision investigation of telomere length widely accessible for laboratory, clinical, and drug discovery efforts and will allow deeper insights into telomere biology.

Fig. 1. Nanopore telomere profiling is accurate and precise.

(A). Schematic depicting nanopore telomere profiling enrichment strategy. Telomeres are tagged with a biotin adapter (TeloTag), enriched by streptavidin pull-down and sequenced. (B). Subtelomere boundary is identified using an algorithm that detects significant deviation from the telomere repeat pattern. (C). Comparison of enriched and non-enriched Telomere Profiling showing fold enrichment with profiling. (D). Southern blot of telomere lengths from 6 individuals of different age. (E). Telomere length measured by Nanopore Telomere Profiling for the same individuals as in C. (total reads = 21,556). The dashed line represents the median telomere length of each distribution. Each point represents a single telomere read. (F). Intra-assay variability: Telomere length from a single donor was measured 14 times on a single flow cell (total reads = 13,256). (G). Inter-assay variability: Telomere length measured from a single donor across 6 different flow cells (total reads = 19,230) (H) Telomere length profiles from the same samples generated in two different laboratories Johns Hopkins (blue), UC Santa Cruz (gold). The difference in telomere length in base pairs is shown at the top.

Fig. 2. Nanopore Telomere Profiling identified population distribution of telomere shortening with age.

(A). Nanopore telomere length profiles for 11 samples selected, age is noted at bottom. Each point is an individual read (total reads = 47624) (B). Prospective Southern of same samples as A. Age of individual noted at bottom. (C). The mean telomere length was determined for 132 individuals aged 0 to 91 (blue dots). Cord blood lengths are shown in purple. The population distribution and confidence intervals for the 90th (blue), the 50th (green line) and 10th percentiles (red) for telomere length in this population are shown using parameters established for FlowFISH (17). (D). For each individual in A. we examined and the reads that fell into the 50^th, 10^th and 1^st percentile for telomere lengths. Red line dotted line indicates 1kb telomere length. (E). Lymphocyte telomere length from FlowFISH data from (16) (gray dots); cord blood is shown with purple dots. The lengths for 15 individuals with IPF were determined by FlowFISH (red). One point represents two individuals who have nearly identical length and are indistinguishable. (F). Nanopore telomere length profiles from the same 15 individuals with IPF are shown plotted against population distribution from C. (Total reads =32457).

Fig. 3. Chromosome end-specific telomere lengths

Violin plots of the distribution of telomere lengths in HG002. Each end is labeled with the chromosome number and p for the short and q for long arms. The haplotypes for each chromosome end are labeled Maternal (M) and Paternal (P) and colored with the same colors for allelic pairs. The mean, 90th and 10th percentile for each distribution are shown with short horizontal black lines in each violin plot. The distribution of all telomeres lengths across all chromosomes ends is at the far left (All) and the dashed line represents this grand mean of all telomeres. The number of reads for each chromosome end is shown at the bottom of each violin plot. Analysis of the means (ANOM) multiple contrast test of each telomere length distribution against the grand mean of all telomere lengths is shown in fig S5D.

Fig. 4. Conserved telomere lengths across 147 individuals

We used the pangenome reference to assign reads to chromosome ends for 147 individuals and compared to where they mapped in three other references. (A). Matrix heatmap shows what fraction of reads that mapped to a given chromosome end in the pangenome (column) and where they map in CHM13 (rows) with a mapq of 60. Light yellow indicates 0% and dark red indicates 100% of reads mapping to the respective chromosome end. (B). As in A. but mapping to the HG002 Maternal reference (C). As in A. but mapping to the HG002 Paternal reference genome. (D). Bar graph showing the fraction of reads that mapped for each chromosome end in the pangenome to the same chromosome end in all three haploid genomes (CHM13, HG002 maternal and HG002 paternal). Colors are the same as in the heatmaps (E). The relative telomere length for each individual and each chromosome end was calculated. The grand mean telomere length for a given individual was subtracted it from the chromosome specific mean telomere length for each chromosome end in that individual. Zero indicates no difference between the chromosome specific mean telomere length and the individual’s grand mean telomere length. Bars represent mean length of a given telomere in all individuals and whiskers represent the standard error of the mean. (F). Same as in E. but for cord blood samples only.