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Native telomere sequencing of individual chromosomes using 3’ end adapter ligation


Date: 19th May 2022

Direct ONT adapter ligation to chromosome ends and base-calling models trained on telomeric repeat sequences enables precise telomere length and methylation analysis in human cells

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Fig. 1 Overview of telomere-sequencing laboratory workflow

ONT adapters are ligated to the ends of telomeres using short telomere adapters

Telomeres consist of repetitive motifs (TTAGGGn) with a single-stranded 3’ G-rich overhang (Fig. 1a). Telomere length is associated with cellular aging and diseases such as cancer. Many cancer cells activate telomerase, or alternative mechanisms, to elongate their telomeres and increase cell survival and propagation. Our approach utilises the 3’ telomeric overhang to ligate an intermediate telomere adapter onto the C-rich strand at the chromosome end (Fig. 1b). The 5’ end of the telomere adapter is complementary to the sequencing adapter and allows the anti sense telomere strand to be sequenced in the 5’ to 3’ direction, from the end of the telomere into the sub-telomeric region.

Fig. 2 Telomere sequencing analysis workflow and comparison of enrichment methods

A customised base-caller identifies and maps enriched telomere sequences

We trained a Bonito base-calling model specifically optimised for telomeric repeats, and filtered reads using a noise-cancelling repeat finder (Fig. 2). The isolated telomeric reads were aligned using minimap2 to the CHM13 v2.0 reference and alternative sub-telomeric assemblies. Alignments to the sub-telomeric regions were used to anchor a read to specific chromosome arms. We compared multiple telomere-enrichment methods using the human cell line HG002 (Fig. 2b). We compared our method to other approaches, including a restriction digestion with AluI and MboI, which do not cut in the telomeres, ONT Cas9-based sequencing using guide RNAs targeted to the sub-telomeric region, and whole genome sequencing.

Fig. 3 Comparing telomere length between two human cell lines

Mapping individual telomere reads provides comparative telomere length measurements

We uniquely mapped reads to the P-and Q-arms of individual chromosomes for two human cell lines, HG002 and U-2 OS (Fig. 3). We were able to assign and anchor reads to 44 telomeres for HG002, and 33 telomeres for U-2 OS. Grey boxes denote chromosomes where we could not find uniquely mapping telomere alignments to the reference. This could be a result of large rearrangements and variation within the telomere/sub-telomere boundaries compared to the reference. The average telomere length in HG002 is 3,900 bp (± 1,627.09 std), while that of U-2 OS is 6,300 bp (± 5,895.10 std). U-2 OS is a bone osteosarcoma cell line known to undergo alternative lengthening of telomeres (ALT), a telomerase-independent mechanism for telomere maintenance.

Fig. 4 Using Remora to call methylation in nanopore sequence data

Analysing methylation patterns in native telomeres using Remora

We used Remora to call CpG methylation and modbamtools to visualise methylation of chromosome 11p of the HG002 and U-2 OS cell lines (Fig. 4). The sub-telomeric region immediately adjacent to the telomere of chromosome 11p in HG002 is hypermethylated, while the sub-telomeric region in U-2 OS is hypomethylated. While the Remora model is accurate within the sub-telomeric regions, Remora is not trained for the repetitive nature of the telomeric regions, and calls low levels of unexpected methylation. The future telomere model will include methylation calls or will be incorporated into the base-caller model.

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