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: Myeloid malignancies are associated with a broad and diverse set of genomic alterations, including SNVs, insertions, deletions and gene fusions. Comprehensive characterization of genetic mutations in hematological disorders currently requires a variety of diagnostic tests and takes multiple days to complete. We developed a fully automated NGS Myeloid Assay that offers an easy to use sample-to-report workflow and the capability for processing up to 8 samples per day. Methods: The Genexus System is comprised of two software linked instruments, the Genexus Purification System and the Genexus Integrated Sequencer. The Genexus Purification System was used to isolate the DNA and RNA from blood or bone marrow samples from precharacterized myeloid samples representative of Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) and from the blood of healthy donors. The Genexus Integrated Sequencer was used to dilute the nucleic acids to optimal concentration and to sequence the samples in replicates with Oncomine Myeloid Genexus v2 Assay. Six DNA and RNA samples were sequenced per run per day along with commercially available analytical controls and a No Template Control. The report was generated by the Genexus Software analysis pipeline optimized to detect different variant types with high sensitivity and specificity. Results: The purification workflows were tested with blood input of 50-400uL for DNA and 50-150uL for RNA. Genexus Purification System extracted and quantified nucleic acids showed input dependent yields. DNA and RNA yields obtained with 50uL sample inputs consistently met the 27.5ng DNA and 15ng RNA minimum requirements for Genexus Integrated Sequencer workflows. DNA libraries had >97% Uniformity of Amplicon Coverage and >95% Target Base Coverage at 350x. The percentage ratio of Mapped Reads for DNA and RNA libraries was approximately 80:20. Detected genetic variations included key hotspots in CEBPA, FLT3, IDH1/2, NPM1, NRAS, RUNX1, and U2AF1 genes that are prevalent in AML and MDS. Genexus Variant Calling results showed high reproducibility and high concordance to the Ion GeneStudio S5 sequencing platform (>95%). The analytical controls, AcroMetrix Oncology Hotspot Control, Seraseq Myeloid Mutation DNA Mix and Seraseq Myeloid Fusion RNA Mix, were sequenced with Sensitivity and PPV >95%. Conclusion: The Genexus System offers an automated sample-to-report workflow with minimal hands-on-time and run results in 30 hours which allows an easy to use solution for next day turnaround time. When used with the Oncomine Myeloid GX v2 Assay, it provides accurate and comprehensive information on diverse mutations including fusions that are relevant to the study of myeloid cancers. For research use only. Not for use in diagnostic procedures. Citation Format: Marina Sedova, Alexy Ongpin, Jennifer Burke, Collyn Seeger, Sarah Brozio, Janice Au-Young, Jiajie Huang, Thilanka Jayaweera, Iris Casuga, Milton Huynh, Fiona Hyland. Fully automated sample-to-report NGS workflow for comprehensive genomic profiling for myeloid neoplasms [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 764.
Next Generation Sequencing technology has enhanced oncology research by enabling the detection of relevant variants for clinical and drug discovery programs. The Oncomine Comprehensive Assay v3, a pan-cancer panel, used with the Ion Torrent Genexus System allows for formalin-fixed paraffin embedded (FFPE) samples to be examined across 161 unique genes in an automated workflow from sample to result in 30hrs. This study demonstrates ≥ 90% accuracy for detecting SNV/Indel/CNV and Fusion variants. The Genexus System consists of two software linked instruments, the Genexus Purification System and the Genexus Integrated Sequencer. The purification system is used first to extract and quantify 6 FFPE sections from human colon and lung tissue for matching DNA & RNA pairs. The nucleic acid output plate from the purification instrument is directly transferred to the sequencer for library preparation and sequencing using the Oncomine Comprehensive Assay v3. Variant calling analysis of 6 DNA & RNA pairs are completed the following day. Results show that the variants were detected with ≥ 90% Sensitivity when compared to the Ion Torrent GeneStudio S5 System as an orthogonal method. All variants report p-values ≤ 10^-5 signifying more confidence that the variant call is correct. The Genexus System is a seamless solution for sample to variant calling results. Citation Format: Iris Casuga, Jason Gioia, Janice Au-Young, Frances Chan, Milton Huynh, Michael Crowe. Rapid and accurate variant calling of FFPE samples with the genexus system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2282.
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