Enspyre: a novel enrichment technology for selected DNA variants using pyrophosphorolysis.

Hybridization capture has been a mainstay of molecular enrichment technologies over the last 15 years, providing robust enrichment of target molecules. However, it typically requires significant sequencing depth to capture rare variants, which limits its efficiency and increases costs, especially in applications like minimal residual disease (MRD) monitoring and early cancer detection. Enspyre (Enrichment by selective pyrophosphorolysis and release) is a novel technology designed to address these limitations by enabling selective enrichment of specific variants prior to sequencing. Here, we present proof-of-concept showing that Enspyre can enrich specific variants by a mean of 35-fold compared to hybridization capture. To demonstrate Enspyre's utility in oncology, we tested serial dilutions of contrived MRD samples using 1800 custom-designed Enspyre bait probes. Our results show that Enspyre accurately detects and quantifies the presence of MRD down to 0.01%-0.001% VAF (variant allele frequency) or 10-100 parts per million (ppm), achieving this sensitivity with approximately 4% of the sequencing reads. Overall, Enspyre has the potential to support applications in rare variant detection, early cancer detection, and therapy selection with reduced sequencing depth, data processing time, and storage requirements.