Combining Highly Multiplexed PCR with Semiconductor-Based Sequencing for Rapid Cancer Genotyping
Combining Highly Multiplexed PCR with Semiconductor-Based Sequencing for Rapid Cancer Genotyping
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DOI：
10.1016/j.jmoldx.2012.09.003
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发表时间：
2013-03-01
影响因子：
4.1
通讯作者：
Corless, Christopher L.
中科院分区：
文献类型：
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作者：
Beadling, Carol;Neff, Tanaya L.;Corless, Christopher L.
临床癌症样本中可操作突变的常规鉴定需求日益增长。基因分型平台必须能快速出结果，并能有效地处理有限的福尔马林固定、石蜡包埋（FFPE）组织样本，这些样本往往产生质量较差的DNA。我们描述了使用一个针对46个癌症基因的190个扩增子的单管多重检测组合对FFPE样本中的DNA进行基于半导体的测序。仅需10纳克的输入DNA，在48小时内就能获得平均2000倍的读取深度，且95%以上的读取是靶向的。一组45个FFPE肿瘤样本的验证集被用于评估检测性能，这些样本包含之前用基于质谱的基因分型平台鉴定出的53个点突变，以及19个4到63个碱基对的插入缺失突变。以8%的突变等位基因比例截断值，我们能够实现点突变检测100%的灵敏度（95%置信区间 = 97.3%到100.0%）和95.1%的特异性（95%置信区间 = 91.8%到98.0%）。通过手动检查比对后的读取，所有插入缺失突变都可见；9个插入缺失突变中的6个……
There is growing demand for routine identification of actionable mutations in clinical cancer specimens. Genotyping platforms must provide rapid turnaround times and work effectively with limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissue specimens that often yield poor quality DNA. We describe semiconductor-based sequencing of DNA from FFPE specimens using a single-tube, multiplexed panel of 190 amplicons targeting 46 cancer genes. With just 10 ng of input DNA, average read depths of 2000x can be obtained in 48 hours, with >95% of the reads on target. A validation set of 45 FFPE tumor specimens containing 53 point mutations previously identified with a mass spectrometry-based genotyping platform, along with 19 indels ranging from 4 to 63 bp, was used to evaluate assay performance. With a mutant allele ratio cutoff of 8%, we were able to achieve 100% sensitivity (95% CI = 97.3% to 100.0%) and 95.1% specificity (95% CI = 91.8% to 98.0%) of point mutation detection. All indels were visible by manual inspection of aligned reads; 6/9 indels