Henzler Christine, Schomaker Matthew, Yang Rendong, Lambert Aaron P, LaRue Rebecca, Kincaid Robyn, Beckman Kenneth, Kemmer Teresa, Wilson Jon, Yohe Sophia, Thyagarajan Bharat, Nelson Andrew C
Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA.
Research Informatics Solutions, Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, MN, USA.
Ann Transl Med. 2018 May;6(9):162. doi: 10.21037/atm.2018.05.07.
Massively parallel, or next-generation, sequencing is a powerful technique for the assessment of somatic genomic alterations in cancer samples. Numerous gene targets can be interrogated simultaneously with a high degree of sensitivity. The clinical standard of care for many advanced solid and hematologic malignancies currently requires mutation analysis of several genes in the front-line setting, making focused next generation sequencing (NGS) assays an effective tool for clinical molecular diagnostic laboratories.
We have utilized an integrated microfluidics circuit (IFC) technology for multiplex PCR-based library preparation coupled with a bioinformatic method designed to enhance indel detection. A parallel low input PCR-based library preparation method was developed for challenging specimens with low DNA yield. Computational data filters were written to optimize analytic sensitivity and specificity for clinically relevant variants.
Minimum sequencing coverage and precision of variant calls were the two primary criteria used to establish minimum DNA mass input onto the IFC. Wet-bench and bioinformatics protocols were modified based on data from the optimization and familiarization process to improve assay performance. The NGS platform was then clinically validated for single nucleotide and indel (up to 93 base pair) variant detection with overall analytic accuracy of 98% (97% sensitivity; 100% specificity) using as little as 3 ng of formalin-fixed, paraffin-embedded DNA or 0.3 ng of unfixed DNA.
We created a targeted clinical NGS assay for common solid and hematologic cancers with high sensitivity, high specificity, and the flexibility to test very limited tissue samples often encountered in routine clinical practice.
大规模平行测序，即新一代测序技术，是评估癌症样本中体细胞基因组改变的一项强大技术。众多基因靶点可同时被高度灵敏地检测。目前，许多晚期实体瘤和血液系统恶性肿瘤的临床标准治疗方案要求在一线治疗中对多个基因进行突变分析，这使得聚焦式新一代测序（NGS）检测成为临床分子诊断实验室的有效工具。
我们利用了一种集成微流控芯片（IFC）技术进行基于多重PCR的文库制备，并结合一种旨在增强插入缺失检测的生物信息学方法。针对DNA产量低的挑战性样本，开发了一种基于低输入量PCR的平行文库制备方法。编写了计算数据过滤器，以优化对临床相关变异的分析灵敏度和特异性。
变异位点检测的最低测序覆盖度和精度是确定输入到IFC上的最低DNA量的两个主要标准。根据优化和熟悉过程中获得的数据，对实验台操作和生物信息学方案进行了修改，以提高检测性能。然后，该NGS平台在临床上得到验证，可用于单核苷酸和插入缺失（长达93个碱基对）变异检测，使用低至3 ng福尔马林固定石蜡包埋DNA或0.3 ng未固定DNA时，总体分析准确率为98%（灵敏度97%；特异性100%）。
我们创建了一种针对常见实体瘤和血液系统癌症的靶向临床NGS检测方法，具有高灵敏度、高特异性，并且能够灵活检测常规临床实践中经常遇到的非常有限的组织样本。
