Detection of Targetable Alterations in Non-small Cell Lung Cancer using Next-generation Sequencing

The success of targeted therapy in non-small cell lung cancer (NSCLC) hinges on the precise identification of driver alterations, including mutations, gene fusions, and amplifications. Next-generation sequencing (NGS) has emerged as a comprehensive molecular diagnostic tool, capable of detecting both known and novel genomic aberrations, providing critical support for personalized NSCLC treatment. However, NGS remains a complex and technically challenging method. Despite its widespread adoption, NGS still faces some challenges, including technical complexity and prolonged turnaround times. Here, the ISO15189-certified NGS workflow implemented in the clinical laboratory is introduced. The standardized protocol encompassed tumor cellularity assessment (≥20 %), DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues (DNA input ≥ 50 ng), automated library preparation, and bioinformatics analysis. By integrating stringent quality control (QC) measures at each step, the workflow ensures high data reliability. Besides, the key innovation in workflow was the automation of NGS library construction. The automated system of NGS library construction included end repair, A-tailing, adapter ligation, hybridization capture, and purification, effectively minimizing human error, enhancing experimental reproducibility, reducing hands-on time, and thus improving efficiency. Together, experience demonstrates that rigorous QC and automated library preparation are essential for maintaining accuracy and scalability in clinical NGS testing. This optimized approach not only ensures compliance with ISO15189 standards but also supports the growing demand for precision oncology in NSCLC management.