Abstract 331: Ion TorrentTM NGS sequencing of the TERT promoter hotspots with Ion AmpliSeqTM HD technology

Abstract

Abstract Introduction: Recurrent mutations C228T and C250T in the TERT gene promoter are prevalent in cancer and serve as important biomarkers for aggressive disease. Ion Torrent™ NGS sequencing, combined with Ion AmpliSeqTM HD technology, enables comprehensive genomic profiling with ultra-high sensitivity for low-frequency variants in cell-free DNA (cf-DNA) and highly heterogeneous solid tumor samples. However, the high GC content (>80%) and the presence of G repeats that are known to adopt a compact stacked three-G-quadruplex conformation in the TERT gene promoter region pose sequencing challenges such as low read depth and strand-bias, i.e. prematurely truncated reads on one strand. Methods: We designed nine AmpliSeqTM HD panels with amplicon sizes of 70 to 104 bp, ideal for cf-DNA and FFPE samples, to target the recurrent TERT promoter hotspots. The designs were tested alone and combined with a larger Ion AmpliSeqTM HD DNA research panel resulting in highly multiplexed libraries. The libraries were used for Ion 540TM chip template preparation on the Ion ChefTM instrument and subsequently sequenced using the Ion GeneStudioTM S5 System. Sequencing data was used to evaluate coverage of the target hotspots by each design for each condition, and additional panels were designed to test single amplicons covering both TERT promoter hotspots. Results: Through performance evaluation, we selected two overlapping amplicons, each effectively amplifying one of the two hotspots. Individually tested, these amplicons yielded approximately 3000 families each from a 10ng gDNA input and when combined into the larger Ion AmpliSeqTM HD DNA research panel, they produced several thousand reads across various libraries, providing molecular coverage of around 200 functional molecules. While capture efficiency was lower compared to TERT amplicon-only libraries, these improved amplicons allow downstream analysis of variants. A single-pool solution (single amplicon/s covering both hotspots) will be explored further using the second batch of amplicons/primers. Conclusions: The optimized amplicons in two pools developed in this study enable the detection of challenging yet clinically significant TERT promoter hotspot mutations using the ultra-sensitive Ion AmpliSeqTM HD technology. For Research Use Only. Not for use in diagnostic procedures. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Citation Format: Anupma Sharma, Guobin Luo, Na Li, Giorgio Pea, Fiona Hyland. Ion TorrentTM NGS sequencing of the TERT promoter hotspots with Ion AmpliSeqTM HD technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 331.