Abstract 221: Development of a rapid, highly sensitive cell-free RNA NGS assay for the detection of gene fusion and splicing variants in cancers

Abstract

Abstract Background: Utility of cell-free nucleic acids (cf-NAs) for early detection and surveillance of tumor represents a new path in personalized medicine. Gene fusions, which usually arise from chromosomal rearrangement, play pivotal roles in numerous human malignancies and have been exploited for disease diagnosis and therapeutics. RNA sequencing, which is not influenced by the complication of introns, is considered superior to DNA sequencing for the identification of fusion genes and splicing variants, and has emerged as a critical tool in cancer liquid biopsy. Methods: We developed a novel approach of preparing strand-specific RNA-Seq libraries. The cDNA produced from the cell-free RNA (cfRNA) samples were labelled with unique molecular identifiers (UMI) during library construction. The libraries were enriched using hybrid capture and the sequencing data were analyzed using Predicine in-house analysis pipeline. To assess the performance of the newly developed cfRNA assay, we compared it with a variety of commercial RNA library kits. Results: The new RNA library construction workflow was streamlined, and it reduces the library preparation time from 2 days to 6 hours with less than 1 h of hands-on time. The yield of the library prepared by the new method was higher than those of the commonly used commercial kits on the market. Sequencing analysis and Droplet Digital PCR (ddPCR) measurement both revealed that our assay was more sensitive in detecting gene fusions and splicing variants in cfRNA than other commercial kits. The assay can reliably detect fusion/splicing variants down to the level of <10 copies of input cfRNA with high specificity. Conclusions: We developed a new RNA library preparation method that produced strand-specific libraries from low input quantities of cfRNA in a fast, simple, and robust manner. This RNA-Seq assay has demonstrated superior performance in known fusion detection and novel fusion discovery in cfRNA. Citation Format: fang liu, xiangying sun, jianrong dong, kemin zhou, yong huang, shidong jia, pan du, Binggang xiang, shujun luo. Development of a rapid, highly sensitive cell-free RNA NGS assay for the detection of gene fusion and splicing variants in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 221.