Enhanced Detection of Novel Low‐Frequency Gene Fusions via High‐Yield Ligation and Multiplexed Enrichment Sequencing

Panel‐based methods are commonly employed for the analysis of novel gene fusions in precision diagnostics and new drug development in cancer. However, these methods are constrained by limitations in ligation yield and the enrichment of novel gene fusions with low variant allele frequencies. In this study, we conducted a pioneering investigation into the stability of double‐stranded adapter DNA, resulting in improved ligation yield and enhanced conversion efficiency. Additionally, we implemented blocker displacement amplification, achieving a remarkable 7‐fold enrichment of novel gene fusions. Leveraging the pre‐enrichment achieved with this approach, we successfully applied it to Nanopore sequencing, enabling ultra‐fast analysis of novel gene fusions within one hour with high sensitivity. This method offers a robust and remarkably sensitive mean of analyzing novel gene fusions, promising the discovery of pivotal biomarkers that can significantly improve cancer diagnostics and the development of new therapeutic strategies. The state of double‐strand stem in adapter imposes effect on ligation strategy and reaction equilibrium. We conducted a pioneering investigation into the stability of double‐stranded adapter DNA and cascaded it with blocker displacement amplification technology, enabling improved ligation yield with enhanced conversion efficiency and selective enrichment in mutations with low variant allele frequency.