Anchored random reverse primer sequencing for quantitative detection of novel gene fusions

Identifying novel gene fusions is critical for cancer diagnosis and drug development. While a few advanced methods have shown the capability to detect gene fusions involving unknown partners, comprehensive detection of gene fusions, especially of those with low copy numbers, remains a challenge. Indeed, most current panel-based sequencing methods fall short in reliability and cost efficiency. Here we present a method for detecting potentially novel gene fusions using anchored random reverse primers (ARRP) during PCR-based library construction, allowing the simultaneous capture of mutations and RNA splicing variants. Furthermore, the combination with blocker displacement amplification technology enables a median of 22-fold allele enrichment for gene fusions, achieving a limit of detection ~10-fold lower than that of current technologies and resulting in an 8-fold cost reduction. Using ARRP-seq, we identify numerous novel fusions in 98 clinical tissue samples, showcasing its diagnostic potential in prostate cancer and capacity for personalized diagnostics in cervical cancer.