Comparison of Next-Generation Sequencing, Quantitative PCR, and Sanger Sequencing for Mutation Profiling of EGFR, KRAS, PIK3CA and BRAF in Clinical Lung Tumors.

BACKGROUND: The clinical application of next-generation sequencing technologies has offered a more comprehensive understanding of the mutational profile of tumor samples. The study was aimed to evaluate the feasibility of the NextDaySeq-Lung panel, which is an NGS-based assay for mutation analysis of key driver genes in lung cancer, in a clinical setting.
METHODS: A total of 138 FFPE samples of non-small cell lung cancer (NSCLC) were examined in parallel with assays developed on the next-generation sequencing (NGS), quantitative PCR (QPCR), and Sanger sequencing (Sanger) platforms for somatic mutations in EGFR, KRAS, PIK3CA, and BRAF. The assays with the three platforms were compared and analyzed.
RESULTS: Compared with Sanger, NGS and QPCR assays have significant higher sensitivity, as Sanger failed to detect variants with mutation rates lower than 15%. Meanwhile, NGS and QPCR assays showed similar analytical sensitivity, specificity, and high concordance. In addition, the NGS assay exhibited advantages over QPCR in providing accurate information of allele sequence and mutation frequency, and detecting non-hotspot mutations.
CONCLUSIONS: We reported the validation of NextDaySeq-Lung panel for mutation analysis in the clinical samples of lung cancer. The NGS assay has significant technical advantages over Sanger and QPCR assays. It shows good potential as a solid molecular diagnostics assay in the clinical setting.