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Timothy Gilpatrick - Cas9 targeted enrichment for nanopore profiling of methylation at known cancer drivers

London Calling 2018

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. We have previously shown the ability to call the methylation status of CpG sites using the signal from nanopore sequencing, and we are aiming to leverage this to profile the methylation status at high sequencing depth of genes known to be involved in tumorigenesis. These efforts have focused on using Cas9-enrichment, sidestepping the relatively high cost/bp of nanopore sequencing while retaining its exquisite sensitivity and long-reads. This allows us to profile signals of methylation over a long range on a single DNA molecule, and by comparing cells with different malignant potential, we can provide new insight into the dynamics of CpG methylation patterns. Specifically, we have targeted the promoter region of the human telomerase gene (hTERT), which is known to have altered methylation patterns that reflect the malignant potential of different cell line. This region is typically difficult to profile with bisulfite amplicon sequencing because of the repetitive and high CG density. Using thyroid cancer cell lines, we have sequenced this region, and identified both methylation level and individual single-read methylation patterns in the samples.

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