Precision Oncology: From Single Mutations to Whole Genomes

FROM SINGLE MUTATIONS TO WHOLE GENOMES | MOLECULAR TECHNOLOGIES SHAPING PRECISION ONCOLOGY DIAGNOSTICS

MOLECULAR TECHNIQUES GUIDING ASSAY DESIGN | tNGS

Hybrid-Capture tNGS Workflow

Amplicon-Based tNGS Workflow

Targeted NGS Breadth & Clinical Depth in One Assay Targeted next-generation sequencing (tNGS) is a technique that bridges the gap between qPCR single-target assays and genome-wide sequencing approaches, offering a high level of multiplexing while maintaining manageable data complexity. Panels typically span 20 to 500 genes and detect a broad range of variant types, including single nucleotide variants (SNVs), small insertions/deletions (indels), gene fusions, and copy number alterations. 15, 16 Many FDA-approved commercial assays, such as those used for solid tumors, hematologic malignancies, and liquid biopsy applications, are now based on tNGS ( Table 2 ). tNGS panels can either employ amplicon-based or hybrid-capture enrichment methods followed by sequencing. Amplicon-based methods rely on qPCR amplification and are typically faster, more cost- efficient, and better suited for small, focused panels, though they have reduced sensitivity for detecting

structural variants and low-complexity regions. 17 In contrast, hybrid-capture methods allow more uniform coverage and improved detection of complex genomic alterations, including gene fusions, copy number variations, and microsatellite instability (MSI), though they require longer workflows and higher DNA input. 18 From a development perspective, tNGS assays provide a scalable solution that can be updated as new biomarkers gain clinical relevance. This makes them well-suited for tumor profiling, therapy selection, and minimal residual disease (MRD) monitoring. However, implementation can be limited by factors such as the need for advanced bioinformatics support, low-quality samples, and longer turnaround times—especially in decentralized or low-resource clinical settings. 14, 19 Despite these challenges, tNGS strikes a valuable balance between genomic breadth, analytical depth, and clinical feasibility—making it a versatile platform for precision oncology applications.

Table 2 . Examples of FDA-Cleared Targeted NGS Assays Used in Clinical Practice