Development of a fully integrated sample-to-report system for a pan-cancer application

Introduction: We have developed a new sample-to-report solution for testing solid tissue cancers using the Ion Torrent System and Software. The assay is designed for research applications from either formalin-fixed, paraffin-embedded (FFPE) solid tumor samples or cell-free total nucleic acid (cfTNA) from liquid biopsy. The new sequencer is a fully automated system requiring minimal hands-on time and touch points for a user to go from nucleic acid to variant calls for somatic variant testing across multiple cancer types. Methods: The assay is a new amplicon-based assay targeting specific somatic variants in 50 genes with coverage for multiple cancer types. The assay uses AmpliSeq HD chemistry capable of distinguishing true sample biologic variants from errors generated during library preparation, templating, and sequencing through incorporation of molecular tags during target amplification. The assay is optimized for use with the Ion Torrent System. With minimal hands-on time (~15 minutes), the user prepares the system with prefilled reagents strips to start a fully automated run that includes library prep, templating, sequencing, variant calling, and a final report if desired. Results: The assay is designed to detect somatic variants in 50 unique genes, where 45 of these genes include substitutions, deletions, and insertions. 14 genes are targeted for copy number variation, and 19 genes are drivers of gene fusions and alternate splice forms. The content has been selected based on published accounts of target actionability and prevalence across multiple cancer types. An example of run setup is shared along with timing (~30min) to input sample information using the Torrent Suite Software. Data are shown from a multiplexed sequencing run using FFPE DNA (10 ng) and RNA (10ng) as input. The automated FFPE run on the Ion Torrent System completed in about 30 hours. Data are also presented from a sequencing run that included multiple liquid biopsy libraries utilizing cell-free total nucleic acid (cfTNA) extracted from plasma. In this run mode, variant calling is optimized for highly sensitive and specific variant calling below 1% allelic frequency for all targeted variant types assay including CNVs, fusions, and alternative splice forms and was accomplished in less than 24 hours. Conclusion: The assay and system enable detection of key oncology variants in 50 genes using both solid tissue and liquid biopsy samples as input. This fully automated solution for oncology research generates variant calls from nucleic acid input in a single 24-hour period with minimal hands-on time and touch-points from the user. This system is not yet released but is in development. Citation Format: Kelli Bramlett, Ru Cao, Jeff Schageman, Yanchun Li, Kris Lea, Priyanka Kshatriya, Amir Marcovitz, Paul Williams, Rasika Sunnadeniya, Oana Lungu, Varun Bagai, Desiree Martin, Khalid Hanif, Jian Gu. Development of a fully integrated sample-to-report system for a pan-cancer application [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B46.