Next-Generation Sequencing technology has enhanced oncology research by enabling the detection of all cancer related variants into one assay for research and drug discovery programs. The Oncomine࣪ Comprehensive Assay v3, a pan-cancer panel, used with the Ion Torrent࣪ Genexus࣪ System allows for formalin-fixed paraffin embedded (FFPE) samples to be examined across 161 unique genes in an automated sample to result workflow in 30hrs. This study demonstrates ≥ 95% Sensitivity and PPV for detecting SNV, Indel, and Copy Number variants of clinical FFPE samples along with fusions. When tested using analytical controls, the Genexus System achieves ≥ 98% Sensitivity for hotspot variants, ≥ 95% for de novo variants, and 100% Sensitivity for fusion variants. The Ion Torrent࣪ Genexus࣪ System is fully automated and consists of two software linked instruments, the Ion Torrent࣪ Genexus࣪ Purification System and the Ion Torrent࣪ Genexus࣪ Integrated Sequencer. For this study, the purification system was used to sequentially extract and quantify DNA & RNA samples from human colon and lung FFPE tumor tissue. The purification instrument provides minimal hands-on-time, ease of use and compatibility with the integrated sequencer. It extracts and quantifies the nucleic acids, records the quantitation values and transfers the nucleic acids to an output plate. The plate is transferred from the purification instrument directly to the sequencer for sample dilution, library preparation, and sequencing using the Oncomine࣪ Comprehensive Assay v3 panel. Variant calling analysis of the DNA & RNA pairs is completed immediately following sequencing. Results show that the variants are detected reproducibly with ≥ 95% Sensitivity and PPV when compared to the Ion GeneStudio࣪ S5 System as an orthogonal method. All variants in the report were correct with p-values ≤ 10-5. Additionally, a subset of the extracted samples was evaluated using Sanger Sequencing to verify hotspot mutations found in samples sequenced on both the Genexus࣪ System and GeneStudio࣪ S5 System. Results show that all expected hotspot variants were detected. In summary, the Ion Torrent࣪ Genexus࣪ System is a reliable and fast turnaround solution for sample-to-variant calling results. When used with the Oncomine࣪ Comprehensive Assay v3 panel, the system provides accurate identification of tumor markers for oncology research. Citation Format: Iris Casuga, Frances Chan, Milton Huynh, Gregory R. Govoni, Kayla Zochowski, Thilanka Jayaweera, Janice Au-Young. Rapid and accurate variant calling of FFPE samples with the Genexus System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2944.
Introduction: The Ion Torrent Genexus System has redefined the genomic profiling paradigm as the first fully-integrated, next-generation sequencing (NGS) research platform to provide an automated sample-to-report workflow with results in a single day. With a purification instrument, an enhanced chip architecture, and downstream reporting, the Genexus System provides a convenient solution to enable oncology research. Here we highlight the oncology research applications and high-throughput NGS capabilities of the Genexus System with Oncomine Comprehensive Assay Plus (OCA Plus), an oncology research panel that can detect variants, fusions, and evaluate key oncology research endpoints. We demonstrate the ability of OCA Plus on Genexus to evaluate tumor mutational burden (TMB), microsatellite instability (MSI), loss of heterozygosity (LOH), and homologous recombination repair deficiency (HRD). Methods: The high-throughput capabilities of the Genexus System enable it to support large oncology research panels such as OCA Plus, which comprises over 13,000 amplicons. The extensive per sample coverage allows for comprehensive DNA and RNA genomic profiling of relevant cancer biomarkers in over 500 genes including detection of over 1,300 fusion isoforms. We utilized high-molecular weight and FFPE samples, reference controls, and orthogonally tested research samples to evaluate DNA variant calling, RNA fusion calling, and key oncology research endpoints, including MSI, LOH, TMB, and HRD. Results: Commercially sourced reference control and research samples were sequenced using OCA Plus on the Genexus System. Sequencing data metrics showed ≥24 million reads per sample, with four samples supported per run. The high-throughput capacity of the Genexus chip architecture results in >95% of amplicons achieving a minimum of 500X coverage with an average coverage uniformity of ≥95% when evaluated across all >13,000 amplicons. Variant calling was assessed using the AcroMetrix Oncology Hotspot Control which has 328 hotspot variants covered by OCA Plus. Variant calling performance showed a hotspot and de novo variant sensitivity and PPV >95%. MSI score and status were assessed using known MSI-High and microsatellite stable (MSS) research samples and FFPE samples of interest. Results show high concordance with data from OCA Plus on GeneStudio. Conclusions: The increased throughput of the Genexus System combined with minimal touchpoints and a rapid turnaround time enables comprehensive genomic profiling for research assays such as OCA Plus where an increased number of sequencing reads leads to greater sensitivity for detecting rare variants and low-level fusion transcripts. Further, accurate characterization of key oncology research endpoints, such as TMB, MSI, LOH, and HRD, allow the Genexus System to accelerate research in the field of oncology. Citation Format: Kayla Zochowski, Dinesh Cyanam, Geoffrey Lowman, Jennifer Kilzer, Sameh El-Difrawy, Yan Zhu, Tanaya Puranik, Alex Phan, Derek Wong, Edith Kwong, Coleen Nemes, Iris Casuga, Frances Chan, Eun Ji Kim, Jianjun Guo, Vinay Mittal, Emily Norris, Shrutii Sarda, Mohit Gupta, Fan Shen, Steven Roman, Gabriel Vargas, Ying Jin, Annie Kraltcheva, Paul Williams, Amneet Gulati, Justin Rigby, Christopher Hoff, Richard Meade, Elaine Wong-Ho, Andrew Wong, Jamsheed Ghadiri, David Garcia-Viramontes, Scott Myrand, Seth Sadis. High-throughput next-generation sequencing research solutions for detection of oncology variants, gene fusion events, and key oncology endpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 42.
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