Clinical Validation of a Next-Generation Sequencing Screen for Mutational Hotspots in 46 Cancer-Related Genes
Clinical Validation of a Next-Generation Sequencing Screen for Mutational Hotspots in 46 Cancer-Related Genes
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DOI：
10.1016/j.jmoldx.2013.05.003
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发表时间：
2013-09-01
影响因子：
4.1
通讯作者：
Luthra, Rajyalakshmi
中科院分区：
文献类型：
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作者：
Singh, Rajesh R.;Patel, Keyur P.;Luthra, Rajyalakshmi
将下一代测序技术转移到临床实验室改进修正案认证的实验室需要进行严格的验证。在本文中，我们使用Ion Torrent AmpliSeq癌症面板和Ion Torrent Personal Genome Machine（IT-PGM）验证了询问46个癌症相关基因中的740个突变热点的下一代测序筛选。使用10纳克FFPE DNA作为模板扩增70个实体瘤样本中46个基因的突变热点区域，包括22个具有已知突变的存档标本和48个用替代测序平台平行测序的标本。在存档标本中，IT-PGM检测到预期的核苷酸取代（n = 29）和6个插入/缺失中的4个;同时，检测到66个变异体。除了单核苷酸取代之外，这些变体通过替代平台进行了确认。对来自两个具有已知突变的癌细胞系的逐渐稀释的DNA进行重复测序，证明了在10%变异频率下对单核苷酸变异的可靠灵敏度，具有高的运行内和运行间重现性。与自动化Ion Torrent OneTouch系统相比，手动文库制备产生相对上级的测序性能。总体而言，具有使用低量FFPE DNA对多个患者样本进行多重和同时测序的能力的IT-PGM平台对于单核苷酸变体突变分析具有特异性和灵敏度，并且可以容易地并入临床实验室进行常规检测。
Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments-certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions (n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using Low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.