Detecting mutations from single DNA molecules is crucial in many fields but challenging. Next-generation sequencing (NGS) affords tremendous throughput but cannot directly sequence double-stranded DNA molecules (‘single duplexes’) to discern the true mutations on both strands. Here we present Concatenating Original Duplex for Error Correction (CODEC), which confers single duplex resolution to NGS. CODEC affords 1,000-fold higher accuracy than NGS, using up to 100-fold fewer reads than duplex sequencing. CODEC revealed mutation frequencies of 2.72 × 10−8 in sperm of a 39-year-old individual, and somatic mutations acquired with age in blood cells. CODEC detected genome-wide, clonal hematopoiesis mutations from single DNA molecules, single mutated duplexes from tumor genomes and liquid biopsies, microsatellite instability with 10-fold greater sensitivity and mutational signatures, and specific tumor mutations with up to 100-fold fewer reads. CODEC enables more precise genetic testing and reveals biologically significant mutations, which are commonly obscured by NGS errors.

Liquid biopsy measurements, such as analysis of circulating tumor DNA (ctDNA) shed by cancer cells, have garnered significant attention for their potential to empower the field of precision oncology. Given that ctDNA tests could enable minimally invasive monitoring and molecular profiling of disease, they are being investigated for use in earlier detection via pan-cancer screening tests, for tracking tumor evolution to inform therapy selection, and for making treatment decisions during minimal residual disease surveillance. However, a typical blood draw carries ultra-low levels of ctDNA (as low as 1.7 copies of the tumor genome in 15mL of blood for a 1cm lung tumor), and this fundamentally limits the sensitivity and the clinical utility of ctDNA-testing in many settings. To push beyond current ctDNA detection limits, we present a first-in-class liquid biopsy priming agent that is given prior to a blood draw to increase the abundance of ctDNA in circulation. Our priming agent consists of liposomes that transiently block the uptake of cell free DNA (cfDNA) by macrophages in the liver, resulting in increased cfDNA available for diagnostic analysis in blood. Using an in vitro 2D assay, we first identified a DSPE-based liposomal formulation that inhibits the uptake of cfDNA by two independent murine macrophage cell lines. Next, we injected our liposomal agent into healthy or tumor-bearing mice and collected blood for further analysis. We found that in healthy mice priming increases the half-life and the recovery of cfDNA from a blood draw, assayed via qPCR. In tumor-bearing mice, the priming agent increases the recovery of ctDNA by up to 60-fold (P = 0.0103) and improves the sensitivity of a ctDNA diagnostic assay from 0% to 75% at low tumor burden. Importantly, cfDNA levels in mice return to baseline within five hours of agent administration and repeated dosing shows no evidence of toxicity. Our priming strategy should be of interest for precision oncology applications, including early detection, longitudinal monitoring of therapeutic response, and surveillance for minimal residual disease. This tumor-agnostic priming agent should also improve the performance of ctDNA analytical techniques other than mutational profiling and may even increase the recovery of cfDNA from other body fluids beyond plasma. Moreover, we believe that this work sets a precedent for the potential development of priming agents for liquid biopsy at large across other analytes. In summary, here we present a first-in-class liquid biopsy priming agent capable of improving the sensitivity and the robustness of ctDNA testing in tumor-bearing mice by modulating liver cfDNA clearance. We envision that further development of liquid biopsy priming agents will signify a major step forward for the successful deployment of precision oncology tools across all stages of cancer management. Citation Format: Carmen Martin-Alonso, Shervin Tabrizi, Kan Xiong, Ahmet Bekdemir Bekdemir, Sahil Patel, Zhenyi An, Timothy Blewett, Sainetra Sridhar, Douglas Shea, Ava Amini, Jesse D. Kirkpatrick, Jin Bae, Eugenia Roberts, Ruolin Liu, Justin Rhoades, Todd Golub, J. Christopher Love, Viktor A. Adalsteinsson, Sangeeta N. Bhatia. A liposomal priming agent increases the sensitivity of liquid biopsies. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR007.