Real-World Technical Hurdles of ctDNA NGS Analysis: Lessons from Clinical Implementation

Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) represents a minimally invasive alternative to conventional tissue biopsies, providing real-time genomic snapshots of heterogeneous tumors from blood draws. This liquid biopsy approach has demonstrated significant utility for early detection, molecular profiling, and monitoring treatment response in cancer patients. However, significant barriers to widespread clinical implementation still remain, such as a lack of standardized methods for ctDNA content quantification and limited variant detection sensitivity at ultra-low frequencies. Herein, we discuss three key improvements: (i) reducing the limit of detection (LoD) from 0.5% to 0.1%, which would increase alteration detection from 50% to approximately 80%; (ii) developing a dynamic LoD approach calibrated to sequencing depth, thereby enhancing result reliability and confidence in clinical interpretation; and (iii) utilizing strategic bioinformatics pipelines with "allowed" and "blocked" lists to enhance accuracy while minimizing false positives. While ctDNA analysis remains approximately 30% less sensitive than tissue-based testing, addressing these limitations through technological advancement and standardization protocols could accelerate integration into routine clinical practice, potentially transforming cancer management while reducing healthcare costs.