A novel Cas9-targeted long-read assay for simultaneous detection of IDH1/2 mutations and clinically relevant MGMT methylation in fresh biopsies of diffuse glioma

Molecular biomarkers provide both diagnostic and prognostic results for patients with diffuse glioma, the most common primary brain tumor in adults. Here, we used a long-read nanopore-based sequencing technique to simultaneously assessIDHmutation status andMGMTmethylation level in 4 human cell lines and 8 fresh human brain tumor biopsies. Currently, these biomarkers are assayed separately, and results can take days to weeks. We demonstrated the use of nanopore Cas9-targeted sequencing (nCATS) to identifyIDH1andIDH2mutations within 36 h and compared this approach against currently used clinical methods. nCATS was also able to simultaneously provide high-resolution evaluation ofMGMTmethylation levels not only at the promoter region, as with currently used methods, but also at CpGs across the proximal promoter region, the entirety of exon 1, and a portion of intron 1. We compared the methylation levels of all CpGs toMGMTexpression in all cell lines and tumors and observed a positive correlation between intron 1 methylation andMGMTexpression. Finally, we identified single nucleotide variants in 3 target loci. This pilot study demonstrates the feasibility of using nCATS as a clinical tool for cancer precision medicine.