Nanopore Sequencing for Targeted Methylation Analysis at Cancer Driver Genes

CpG methylation in the mammalian genome is known to alter the binding of transcriptional regulatory factors, and through this activity mediate changes in chromatin structure and gene expression. Nanopore sequencing is sensitive to the methylation status of CpG sites as revealed by modulation to the raw electrical signal; we have previously developed a method to use this data to profile patterns of methylation over a long range on native DNA molecules.

The yield from nanopore sequencing is still relatively low (~5-10Gb per flowcell) as compared to the size of the human genome, suggesting the need for a targeted sequencing strategies. There exist targeted approaches using hybridization capture or size selection after excision, yet a need persists for methods providing higher coverage at regions of interest with limited input DNA. Here, we use the CRISPR/Cas9 system to introduce breaks for ligating nanopore sequencing adaptors, and subsequently evaluate methylation at oncogene promoters, decreasing the relatively high cost/bp of nanopore sequencing while retaining its exquisite sensitivity and long-reads.

Specifically, we have targeted the promoter region of the human telomerase gene (hTERT) in thyroid cancer cell lines, as activation of this gene plays a critical role in the immortalization of cells, and methylation of the TERT promoter is known to affect transcription factor binding and gene expression. This region is generally difficult to analyze because of its repetitive nature and high CpG density. Examination of the TERT promoter with nanopore sequencing showed methylation patterns that closely track those seen with illumina bisulfiite amplicon sequencing, but adds allele-specific methylation level information and phased methylation patterns. Our method provides a valuable and efficient approach with applications in clinical and research labs--using nanopore sequencing for analysis of DNA methylation, evaluation of structural variants, and assignment of reads to allele of origin.