Targeted nanopore sequencing with Cas9 for studies of methylation, structural variants and mutations
About Timothy Gilpatrick
Timothy Gilpatrick is a current MD/PhD student at Johns Hopkins University in Baltimore, USA. He is doing his PhD work in the lab of Winston Timp, where his studies have centred on the use of nanopore sequencing to study cancer epigenetics and structural variation. He received his BSc in Biochemistry from the University of Delaware, working in a protein structure lab to characterize the role of lipoprotein-associated enzymes in atherosclerosis. Prior to starting his graduate studies, he worked as a research fellow at the National Institutes of Health, examining how microRNAs regulate histone modifications in embryonic stem cells.
There is an existing need for clinical tools that can be used to rapidly assess genomic variants and epigenetic changes at medically relevant genes. We have been using the CRISPR/Cas9 system for target-enrichment nanopore sequencing. We show the ability of this method to generate greater than 200X average coverage at 10 genomic loci (mean size 18kb) with a single MinION flow cell. We demonstrate that this high coverage data enables us to (1) profile DNA methylation patterns at cancer driver genes, (2) detect structural variations at known hot spots, and (3) survey for the presence of single nucleotide mutations. We demonstrate applications of this technique by examining the well-characterized GM12878 cell line as well as three breast cell lines (MCF-10A, MCF-7, MDA-MB-231) with varying tumorigenic potential as a model for cancer.