Abstract 337: Balanced-strand sequencing for highly efficient duplex variant calling in circulating tumor DNA
Authors: Widman Adam , Cheng Alexandre P. , Sossin Aaron , Rajagopalan Srinivas , Al Assaad Majd , Deochand Sunil , Steinsnyder Zoe , Manaa Dina , Marton Melissa , Reeves Catherine , Rusinek Itai , Meiri Eti , Barad Omer , Shipony Zohar , Gilad Shlomit , Jaimovich Ariel , Sigouros Michael , Manohar Jyothi , King Abigail , Wilkes David , Otilano John , Elemento Olivier , Faltas Bishoy M. , Mosquera Juan M. , Landau Dan A. 
Published: March 2024

Abstract
Several approaches for the detection of circulating tumor DNA (ctDNA) are anchored in the accurate identification of cancer-specific single nucleotide variants (SNVs) in plasma. Such approaches are limited in ultra-low tumor fraction (TF) contexts, such as early cancer detection or the identification of postoperative minimal residual disease, where ctDNA SNVs are difficult to distinguish against background sequencing error. ctDNA detection in these critical low TF contexts is further limited by the sparse amount of cell free DNA (cfDNA) in plasma (Zviran et al., 2020). To reduce single-stranded artifact and base calling errors, duplex sequencing can be used to jointly analyze corresponding Watson and Crick strands to detect true, double-stranded variants. Recent work has demonstrated that duplex sequencing can decrease error rates to 10−7 (Hoang et al., 2016) and can be deployed in the whole-genome sequencing (WGS) setting (Cheng et al., 2023). A significant limitation for existing duplex techniques is the need to analytically pair matching DNA strands through exhaustive sequencing, resulting in inefficient use of scarce input material, particularly in plasma cfDNA. These techniques typically capture 1-5% of sequenced molecules, restricting duplex coverage depth. We present balanced-strand sequencing, a PCR-free approach that leverages the hydrogen bonds of complementary DNA to carry matched native strands directly into sequencing, enabling massively scalable duplex sequencing. The approach relies on Ultima Genomics sequencing, where DNA denaturation is not required prior to clonal amplification. In balanced strand sequencing, double-stranded DNA is partitioned and clonally amplified on sequencing beads through emulsion-PCR. Each double-stranded DNA molecule contributes to a single sequencing read, allowing for a linear increase in duplex recovery with increasing sequencing depth. The approach does not require redundant sequencing and therefore maximizes unique molecule throughput. We applied balanced strand sequencing to cfDNA, input as low as 5 ng, and achieved 7-35x whole-genome duplex coverage, roughly 20-fold higher than our prior duplex WGS. We further developed a machine-learning guided single read mutation calling framework, which when combined with duplex error suppression, reduced error rates to 10−7 and enabled accurate ctDNA detection at concentrations &lt;10−5. Use of our analysis framework in APOBEC-mutant bladder cancer plasma samples allowed us to identify APOBEC signatures in plasma and platinum exposure signatures indicative of prior chemotherapy treatment. Altogether, balanced strand sequencing radically enhanced duplex efficiency and has broad applications across cancer. In cfDNA applications, balanced strand sequencing enhances error-correction in tumor-informed ctDNA detection and paves the way for non-tumor informed WGS ctDNA detection.
Citation Format: Adam Widman, Alexandre P. Cheng, Aaron Sossin, Srinivas Rajagopalan, Majd Al Assaad, Sunil Deochand, Zoe Steinsnyder, Dina Manaa, Melissa Marton, Catherine Reeves, Itai Rusinek, Eti Meiri, Omer Barad, Zohar Shipony, Shlomit Gilad, Ariel Jaimovich, Michael Sigouros, Jyothi Manohar, Abigail King, David Wilkes, John Otilano, Olivier Elemento, Bishoy M. Faltas, Juan M. Mosquera, Dan A. Landau. Balanced-strand sequencing for highly efficient duplex variant calling in circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 337.