No abstract

Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via ‘End Repair/dA-Tailing,’ may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7–17% and 32–57% of interior ‘duplex base pairs’ from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles’ heel in sequencing and offers a solution to restore high accuracy.

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.

Accurate DNA sequencing is crucial in biomedicine. Underlying the most accurate methods is the assumption that a mutation is true if altered bases are present on both strands of the DNA duplex. We now show that this assumption can be wrong. We establish that current methods to prepare DNA for sequencing, via End Repair/dA-Tailing, may substantially resynthesize strands, leading amplifiable lesions or alterations on one strand to become indiscernible from true mutations on both strands. Indeed, we discovered that 7-17% and 32-57% of interior duplex base pairs from cell-free DNA and formalin-fixed tumor biopsies, respectively, could be resynthesized in vitro and potentially introduce false mutations. To address this, we present Duplex-Repair, and show that it limits interior duplex base pair resynthesis by 8- to 464-fold, rescues the impact of induced DNA damage, and affords up to 8.9-fold more accurate duplex sequencing. Our study uncovers a major Achilles heel in sequencing and offers a solution to restore high accuracy.

Liquid biopsies using cell-free DNA (cfDNA) enable non-invasive detection and characterization of disease. Advances in sequencing methods have significantly improved the performance of liquid biopsies. Yet, despite these advances, sensitivity remains a fundamental challenge. In oncology, circulating tumor DNA (ctDNA) screening tests only detect 20-40% of stage I tumors and tests for minimal residual disease have only 25-50% sensitivity after surgery. The major barrier to better sensitivity is the intrinsic low level of ctDNA in plasma. Physical absence of tumor DNA molecules in a blood draw from a patient with low disease burden will result in a negative test, no matter the sensitivity of the ex vivo detection platform. To overcome this barrier, here we report a first-in-class intravenous DNA-binding priming agent that is given 2 hours prior to a blood draw to recover more ctDNA, boosting the detection of tumor mutations in plasma by 19-fold and increasing sensitivity from 6% to 84%. Given the rapid clearance of cfDNA from circulation, we reasoned that a priming agent that could bind and protect cfDNA from clearance could increase the tumor DNA recovered from plasma. We selected monoclonal antibodies (mAbs) as the class of molecules to use as cfDNA protectors given their persistence in circulation and ease of engineering. We identify a mAb that binds double-stranded DNA (dsDNA) and find on electrophoretic mobility shift assays that it binds both free and histone-bound dsDNA, the constituent components of cfDNA. We then demonstrate that this mAb can delay the clearance of dsDNA from plasma in vivo through co-injection of the mAb with free- and histone-bound dsDNA in mice. We further identify interactions with Fc-gamma-receptors as a key mediator of early clearance of dsDNA bound to the priming mAb. To address this early clearance and limit potential immune interactions, we engineer the mAb to abrogate its Fc effector function. The engineered variant decreases clearance of injected dsDNA by over 150-fold at one hour post-injection compared to dsDNA alone. We next evaluate the effect of our priming mAb on cancer detection. We use a targeted panel against 1,822 mutations in the MC26 murine colon carcinoma cell line to detect tumor mutations in the plasma of tumor bearing mice. The priming mAb results in 19-fold higher recovery of tumor DNA molecules compared to a control mAb. This improved recovery leads to detection of 77% of targeted sites in plasma compared to only 15% in the control group. In sensitivity analyses, higher recovery of mutant molecules improves sensitivity for cancer detection from 6% to 84% at 0.001% tumor fraction. In summary, we demonstrate an approach to overcome a key barrier in liquid biopsies. We envision that similar to contrast agents in clinical imaging, priming agents could significantly boost the diagnostic sensitivity of liquid biopsies and enable further applications across biomedicine. Citation Format: Shervin Tabrizi, Carmen Martin-Alonso, Kan Xiong, Timothy Blewett, Sainetra Sridhar, Zhenyi An, Sahil Patel, Sergio Rodriguez-Aponte, Christopher Naranjo, Douglas Shea, Todd Golub, Sangeeta N. Bhatia, Viktor A. Adalsteinsson, J. Christopher Love. A DNA-binding priming agent protects cell-free DNA and improves the sensitivity of liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3371.