Targeted next-generation sequencing techniques: From primer design to sophisticated library construction

Targeted Next-Generation Sequencing (tNGS) represents a cornerstone technology in modern clinical diagnostics and biological research. Widely adopted by clinical laboratories, tNGS provides a robust and cost-effective alternative to whole-genome sequencing. Despite its extensive applications, the most technically complex and variable stage of the tNGS workflow remains target enrichment, a critical step that ensures target molecules are present at sufficiently high concentrations relative to background sequences. Enrichment methodologies are broadly classified into PCR-based and hybridization-based strategies, each offering distinct advantages and limitations. This review critically examines both approaches within the context of the computational challenges inherent to tNGS assay design, and surveys the full spectrum of established and emerging enrichment techniques, highlighting their suitability across different areas of molecular research and clinical diagnostics. The use of non‑canonical nitrogenous bases, circularization of linear DNA fragments, unique molecular identifiers (UMIs), and partially double‑stranded primers are among the many sophisticated approaches described in the review. We hope this review will contribute to accelerating the development of diagnostic strategies with targeted next-generation sequencing in the rapidly evolving field of molecular diagnostics.