Pillar Biosciences Inc., Massachusetts, United States
Introduction: Detection of variants in cell-free DNA (cfDNA), particularly those at exceptionally low variant allele frequency (VAF), is important for early-stage treatment decision, therapy monitoring, recurrence detection, and improving survival for cancer patients. However, adoption of this technology is limited by challenges such as insufficient cfDNA amount, distinguishing real signals from noise, lack of kitted Liquid Biopsy (LBx) panels for local NGS (Next Generation Sequencing) testing, and cost effectiveness. To address these challenges, we developed oncoReveal™ Core LBx, a cost-effective, unique molecular identifier (UMI) free, 446 amplicon research-use-only (RUO) panel and assessed its performance in calling low-frequency somatic variants. We verified the panel’s efficacy on both clinical and contrived samples.
Methods: Standard reference control samples from Seraseq® were used at VAFs ranging from 0.1-5%, at 10ng and 30ng DNA input, for a total of 37 replicates. cfDNA was extracted from plasma samples (8 - 30ng) from 16 patients, 2 healthy donors, 2 standard reference controls and 1 wild-type control sample. NGS libraries were prepared using the oncoReveal™ Core LBx panel and sequenced on Illumina’s NextSeq™ 550 platform, targeting an average of 30M paired-end reads per sample and variants were called using Pillar’s PiVAT® (Pillar Biosciences Variant Analysis Toolkit).
Results: We were able to detect low-frequency variants, including in samples with low DNA input ( <10ng). We detected 88% and 96% of the expected variants in 0.25% VAF at DNA inputs of 10ng and 30ng, respectively. High-priority variants with known drug response or pathogenicity were detected down to 0.1% VAF. All variants were detected in higher VAF samples. In negative samples, we had >99.99% per-position specificity.
Conclusion: We demonstrate here a low-cost, kitted, amplicon-based (RUO) liquid biopsy panel that can detect clinically significant, high priority variants down to 0.1% VAF, without the use of UMIs. The panel can be run at scale on the mid-throughput Illumina NextSeq 550 NGS platform, enabling greater opportunity for laboratories to perform liquid biopsy-based tumor profiling within their own laboratories. The panel was able to amplify and detect variants even in samples with limited DNA input <10ng. Further, the panel’s performance was not affected by the choice of extraction strategy used.
We are developing a fully automated process for our oncoReveal™ Core LBx on the Biomek NGeniuS instrument (Beckman Coulter) to prepare 4 to 24 libraries with only ~1 hour upfront hands-on time and 6 hours of walkaway time from input DNAs to final libraries. The automated Pillar ORST22 assay on Biomek NGeniuS system produced high quality and robust results with minimal hands-on time and human intervention. Together with the easy-to-use setup, it could substantially reduce the lab operation burden.