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 assess IDH mutation status and MGMT methylation 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 identify IDH1 and IDH2 mutations within 36 h and compared this approach against currently used clinical methods. nCATS was also able to simultaneously provide high-resolution evaluation of MGMT methylation 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 to MGMT expression in all cell lines and tumors and observed a positive correlation between intron 1 methylation and MGMT expression. 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.

中文翻译：

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一种新型的靶向 Cas9 的长读长检测方法，用于同时检测弥漫性胶质瘤新鲜活检组织中的 IDH1/2 突变和临床相关的 MGMT 甲基化。

分子生物标志物为弥漫性神经胶质瘤（成人最常见的原发性脑肿瘤）患者提供诊断和预后结果。在这里，我们使用基于长读长纳米孔的测序技术来同时评估 4 个人类细胞系和 8 个新鲜人类脑肿瘤活检组织中的 IDH 突变状态和 MGMT 甲基化水平。目前，这些生物标志物是单独分析的，结果可能需要几天到几周的时间。我们展示了使用纳米孔 Cas9 靶向测序 (nCATS) 在 36 小时内识别 IDH1 和 IDH2 突变，并将这种方法与目前使用的临床方法进行了比较。nCATS 还能够同时提供对 MGMT 甲基化水平的高分辨率评估，不仅在启动子区域，与目前使用的方法一样，而且在跨近端启动子区域的 CpG，整个外显子 1，和一部分内含子 1。我们比较了所有细胞系和肿瘤中所有 CpG 的甲基化水平与 MGMT 表达，并观察到内含子 1 甲基化与 MGMT 表达之间呈正相关。最后，我们在 3 个目标基因座中鉴定了单核苷酸变体。这项试点研究证明了使用 nCATS 作为癌症精准医学临床工具的可行性。