Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer.
Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer.
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DOI：
10.1093/dnares/dsx027
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发表时间：
2017-12-01
期刊：
影响因子：
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通讯作者：
Suzuki Y
中科院分区：
文献类型：
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作者：
Suzuki A;Suzuki M;Mizushima-Sugano J;Frith MC;Makalowski W;Kohno T;Sugano S;Tsuchihara K;Suzuki Y
在这里，我们采用了cDNA扩增子测序使用长读便携式测序仪，MinION，表征癌症相关基因，即EGFR，KRAS，NRAS和NF 1的各种类型的突变。对于纯合子SNV，准确率和召回率分别为87.5%和91.3%。对于先前报道的热点突变，准确率和召回率达到100%。还检测到EML 4-ALK、CCDC 6-RET和其他五种基因融合体的精确连接。利用长读序测序技术，我们对EGFR突变进行了定相，并阐明了抗肿瘤药物敏感和耐药突变的突变等位基因背景，为治疗方案的选择提供了有用的信息。在H1975细胞中，72%的读段具有L 858 R和T790 M突变，22%的读段不具有突变。为了确保在潜在的低癌细胞群体中可以满足临床要求，我们进一步对EGFR突变的模板进行了连续稀释分析。根据测序数据的产量和质量，可以检测到百分之几的突变等位基因。最后，我们在8个临床样本的突变基因型的特点。这种方法可能是一种方便的基于长读序测序的分析方法，因此可能会改变目前用于癌症基因组测序的方法。
Here, we employed cDNA amplicon sequencing using a long-read portable sequencer, MinION, to characterize various types of mutations in cancer-related genes, namely, EGFR, KRAS, NRAS and NF1. For homozygous SNVs, the precision and recall rates were 87.5% and 91.3%, respectively. For previously reported hotspot mutations, the precision and recall rates reached 100%. The precise junctions of EML4-ALK, CCDC6-RET and five other gene fusions were also detected. Taking advantages of long-read sequencing, we conducted phasing of EGFR mutations and elucidated the mutational allelic backgrounds of anti-tumor drug-sensitive and resistant mutations, which could provide useful information for selecting therapeutic approaches. In the H1975 cells, 72% of the reads harbored both L858R and T790M mutations, and 22% of the reads harbored neither mutation. To ensure that the clinical requirements can be met in potentially low cancer cell populations, we further conducted a serial dilution analysis of the template for EGFR mutations. Several percent of the mutant alleles could be detected depending on the yield and quality of the sequencing data. Finally, we characterized the mutation genotypes in eight clinical samples. This method could be a convenient long-read sequencing-based analytical approach and thus may change the current approaches used for cancer genome sequencing.