Abstract A037: Improved liquid biopsy assay performance using novel highly accurate sequencing-by-binding (SBB) technology
Authors: Sockell Alexandra , Nasko Dan , Kim Young , Kim Kristi , Korlach Jonas 
Published: November 2024

Abstract
Liquid biopsy is revolutionizing the field of early cancer detection research through non-invasive detection of tumor DNA in the blood. However, existing liquid biopsy assays are limited in their sensitivity for ctDNA detection at low variant allele frequencies (VAFs), with most relying on extreme sequencing depth and computational error correction to separate the true ctDNA signal from background errors. This limitation is particularly problematic in the area of early cancer detection and minimal residual disease monitoring, in which expected ctDNA allele frequencies are extremely low. Novel strategies are therefore needed to help improve liquid biopsy assay sensitivity and reduce per-sample sequencing requirements. Here we describe PacBio’s application of the Onso short-read sequencing system to enable detection of ctDNA at low VAFs using the SeraCare Complete ctDNA Mutation Mix reference standard. The Onso system makes use of a novel sequencing by binding (SBB) method to achieve 10- to 100-fold greater quality scores, with ≥90% of reads at Q40 or above. We performed targeted capture and sequencing of libraries prepared from the SeraCare reference mix diluted into WT human DNA at the following VAFs: 0.00% (WT), 0.05%, 0.10%, and 0.25%, and compared the sensitivity at each VAF for SBB compared to a competitor method using sequencing by synthesis (SBS) at varying sequencing depths. At equivalent sequencing depth (12,000X) we observed superior recall for low frequency variants (VAF = 0.05%, 0.1%) in the reference sample for Onso compared to SBS. Furthermore, SBB was able to achieve comparable sensitivity results to SBS using four- fold less sequencing (6,000X vs. 24,000X), and without the use of computational error correction. Combining SBB with error correction methods boosted sensitivity even further, suggesting an additive value for these technologies. Comparison of false positive variant calls in the WT samples showed a dramatic decrease in false positives for Onso (n=2) compared to SBS (n=49) across 10 subsampling replicates, and observed allele frequencies for Onso were closer to the expected values. In conclusion, Onso achieved superior sensitivity for ctDNA detection at compared to SBS at equivalent sequencing depths, and achieved similar performance with four- fold less sequencing and without the use of error correction. This improvement was due in part to the reduction in false positive variant calling in the WT sample, which lowers the limit of detection to maximize variant detection while maintaining high specificity. Taken together, our results demonstrate the potential of SBB to improve upon existing methods of liquid biopsy and better enable research on early cancer detection and minimal residual disease.
Citation Format: Alexandra Sockell, Dan Nasko, Young Kim, Kristi Kim, Jonas Korlach. Improved liquid biopsy assay performance using novel highly accurate sequencing-by-binding (SBB) technology [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A037.