Fig. 1

Mutation and methylation assessments with well-characterized samples was used to develop nCATS workflow. a, Genotyping of IDH1 wild type (purchased), IDH2 wild type (purchased), IDH2 R172K mutation (purchased), and IDH1 R132G mutation (fresh biopsy sample). Exon 4 of IDH1 and IDH2 were PCR amplified and sequenced with nanopore technology. Nanopolish correctly genotyped all samples. b, Observed and expected CpG methylation percentage detected on methylated and unmethylated DNA standards. Standards that were 100% methylated or 0% methylated on CpGs were sequenced, and methylation calling was performed with Nanopolish. Data were generated for 10, 25, 50%, or 75% methylated CpGs by randomly sampling reads from each standard; at ≥20 depth coverage (20X), methylation levels of 0, 25, 50, 75, and 100% could be distinguished. Data represent the median, with 25th and 75th percentiles. Pairwise t-test with Bonferroni correction **** P < 0.0001. Thus, 20X was used as the theoretical limit of detection in this study. c, Guide RNA (crRNA) for 3 target loci (MGMT, IDH1, and IDH2) were designed and used for nanopore Cas9-targeted sequencing (nCATS) with the MinION device. Various types of sample were used for testing the feasibility of nCATS to assay methylation and mutations. GBM, glioblastoma; TMZ, temozolomide. d, Median coverage of each loci for 10 samples