Sussman Robyn T, Shaffer Sydney, Azzato Elizabeth M, DeSloover Daniel, Farooqi Midhat S, Meyer Anders, Lieberman David B, Bigdeli Ashkan, Paolillo Carmela, Ganapathy Karthik, Sukhadia Shrey, Rosenbaum Jason N, Daber Robert D, Morrissette Jennifer J D
Hospital of the University of Pennsylvania, Division of Precision and Computational Diagnostics, Department of Pathology & Laboratory Medicine, 3020 Market Street, Suite 220, Philadelphia, PA 19104, United States.
Hospital of the University of Pennsylvania, Division of Precision and Computational Diagnostics, Department of Pathology & Laboratory Medicine, 3020 Market Street, Suite 220, Philadelphia, PA 19104, United States.
Cancer Genet. 2018 Dec;228-229:55-63. doi: 10.1016/j.cancergen.2018.08.004. Epub 2018 Sep 19.
One caveat of next-generation sequencing (NGS)-based clinical oncology testing is the high amount of input DNA required. We sought to develop a focused NGS panel that could capture hotspot regions in relevant genes requiring 0.5-10 ng input DNA. The resulting Penn Precision Panel (PPP) targeted 20 genes containing clinically significant variants relevant to many cancers. One hundred twenty-three samples were analyzed, including 83 solid tumor specimens derived from FFPE. Various input quantities of DNA (0.5-10 ng) were amplified with content-specific PCR primer pools, then sequenced on a MiSeq instrument (Illumina, Inc.) via paired-end, 2 × 186 base pair reads to an average read depth of greater than 6500x. Variants were detected using an in-house analysis pipeline. Clinical sensitivity and specificity were assessed using results from our previously validated solid tumor NGS panel; sensitivity of the PPP is 96.75% (387/400 variants) and specificity is 99.9% (8427/8428 base pairs). Variant allele frequencies (VAFs) are highly concordant across both assays (r = 0.98 p < 0.0001). The PPP is a robust, clinically validated test optimized for low-yield solid tumor specimens, capturing a high percentage of clinically relevant variants found by larger commercially available NGS panels while using only 0.5-10 ng of input DNA.
基于新一代测序（NGS）的临床肿瘤学检测的一个注意事项是所需的输入DNA量很高。我们试图开发一种聚焦的NGS检测板，它可以捕获相关基因中的热点区域，所需的输入DNA量为0.5 - 10 ng。最终得到的宾夕法尼亚精准检测板（PPP）针对20个包含与多种癌症相关的临床显著变异的基因。分析了123个样本，包括83个源自福尔马林固定石蜡包埋（FFPE）的实体瘤标本。用针对特定内容的PCR引物池对不同输入量的DNA（0.5 - 10 ng）进行扩增，然后在MiSeq仪器（Illumina公司）上通过双端2×186碱基对读数进行测序，平均读深度大于6500倍。使用内部分析流程检测变异。使用我们之前验证过的实体瘤NGS检测板的结果评估临床敏感性和特异性；PPP的敏感性为96.75%（387/400个变异），特异性为99.9%（8427/8428个碱基对）。两种检测方法的变异等位基因频率（VAF）高度一致（r = 0.98，p < 0.0001）。PPP是一种经过临床验证的强大检测方法，针对低产量实体瘤标本进行了优化，在仅使用0.5 - 10 ng输入DNA的情况下，能够捕获较大的市售NGS检测板所发现的高比例临床相关变异。
