Same-day genomic and epigenomic diagnosis of brain tumors using real-time nanopore sequencing
Same-day genomic and epigenomic diagnosis of brain tumors using real-time nanopore sequencing
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DOI：
10.1007/s00401-017-1743-5
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发表时间：
2017-11-01
影响因子：
12.7
通讯作者：
Idbaih, Ahmed
中科院分区：
文献类型：
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作者：
Euskirchen, Philipp;Bielle, Franck;Idbaih, Ahmed
癌症分子分类已经进入临床常规，为诊断、预后和治疗决策提供信息。同时，新的肿瘤实体已被确定，不能确定组织学。对于中枢神经系统肿瘤，目前世界卫生组织的分类明确要求进行分子检测，例如1p/19q编码或IDH突变，以进行综合的组织分子诊断。然而，目前需要大量复杂的技术来评估不同的基因组和表观基因组变化，并且周转时间在几周的范围内，这使得标准化和广泛实施变得困难，并阻碍了及时决策。在这里，我们探索了用于癌症多模式和快速分子诊断的口袋大小的纳米孔测序装置的潜力。使用低通全基因组测序，在同一测序运行中同时从天然肿瘤DNA中生成拷贝数（CN）和甲基化谱。利用深度扩增子测序技术鉴定了IDH1、IDH2、TP53、H3F3A和TERT启动子区域的单核苷酸变异。纳米孔测序在6小时内获得了类似于0.1X的基因组覆盖率，所得到的CN和表观遗传谱与匹配的微阵列数据具有良好的相关性。诊断相关的改变，如1p/19q编码，和病灶放大可以重现。使用特设随机森林，我们可以进行监督的泛癌症分类，以区分胶质瘤、髓母细胞瘤和不同原发部位的脑转移。IDH1、IDH2和H3F3A的单核苷酸变异在测序的几分钟内通过深度扩增子测序被鉴定出来。TP53和TERT启动子突变检测表明，全基因和富gc区测序是可行的。纳米孔测序允许当天检测结构变异，点突变，甲基化分析使用单个设备，可忽略不计的资本成本。在诊断时间和所需的实验室设备和专业知识方面，它优于基于杂交的和当前的测序技术，旨在为每位癌症患者提供精准医疗，即使在资源有限的环境中也是如此。
Molecular classification of cancer has entered clinical routine to inform diagnosis, prognosis, and treatment decisions. At the same time, new tumor entities have been identified that cannot be defined histologically. For central nervous system tumors, the current World Health Organization classification explicitly demands molecular testing, e.g., for 1p/19q-codeletion or IDH mutations, to make an integrated histomolecular diagnosis. However, a plethora of sophisticated technologies is currently needed to assess different genomic and epigenomic alterations and turnaround times are in the range of weeks, which makes standardized and widespread implementation difficult and hinders timely decision making. Here, we explored the potential of a pocket-size nanopore sequencing device for multimodal and rapid molecular diagnostics of cancer. Low-pass whole genome sequencing was used to simultaneously generate copy number (CN) and methylation profiles from native tumor DNA in the same sequencing run. Single nucleotide variants in IDH1, IDH2, TP53, H3F3A, and the TERT promoter region were identified using deep amplicon sequencing. Nanopore sequencing yielded similar to 0.1X genome coverage within 6 h and resulting CN and epigenetic profiles correlated well with matched microarray data. Diagnostically relevant alterations, such as 1p/19q codeletion, and focal amplifications could be recapitulated. Using ad hoc random forests, we could perform supervised pan-cancer classification to distinguish gliomas, medulloblastomas, and brain metastases of different primary sites. Single nucleotide variants in IDH1, IDH2, and H3F3A were identified using deep amplicon sequencing within minutes of sequencing. Detection of TP53 and TERT promoter mutations shows that sequencing of entire genes and GC-rich regions is feasible. Nanopore sequencing allows same-day detection of structural variants, point mutations, and methylation profiling using a single device with negligible capital cost. It outperforms hybridization-based and current sequencing technologies with respect to time to diagnosis and required laboratory equipment and expertise, aiming to make precision medicine possible for every cancer patient, even in resource-restricted settings.