Abstract 59: Optimization of high-throughput, high-depth, targeted single-cell DNA sequencing to pancreatic ductal adenocarcinoma

Abstract

Abstract Purpose of study: Despite insights gained by bulk DNA sequencing of cancer it remains challenging to resolve “mixed signals”, i.e. the admixture of normal and tumor cells, and/or of distinct tumor subclones. Bulk sequencing of pancreatic ductal adenocarcinoma (PDAC) has been particularly problematic due to the high stromal content and resulting low tumor cellularity. Strategies to account for this have included laser capture microdissection yet this is a laborious process not amenable to high-throughput pipelines. We sought to develop and apply a high-throughput, high-depth, targeted single-cell DNA sequencing (scDNA-seq) method to account for these issues, including the ability to extract high quality genomic information from low purity and archival samples. Experimental procedures: Bulk whole exome sequencing (WES) was performed on 29 biologically distinct samples. For single cell sequencing, we developed a custom panel containing 186 amplicons covering 93 genes that represent the most common germline and somatic targets reported in PDAC. Based on a commercially available system that enables automatic enzymatic and mechanical tissue disruption with integrated fluidic processes, we optimized a nuclei extraction workflow from frozen tissues that is highly compatible with downstream microdroplet-based single-cell encapsulation and library preparation. With it we generated scDNA-seq data from archival tissues of 15 PDAC patients at varying stages of the disease. Results were compared to that found in matched bulk sequencing data. Summary of new, unpublished data and conclusions: 42 samples were analyzed by single cell sequencing. Our nuclei extraction workflow generated on average 2867 single cell libraries per sample at >80X read depth. The single-cell results aligned well with matched bulk data in terms of the detection of key genetic variants and their variant allele frequency (VAF). We also identified additional driver variants not seen by WES, some with direct clinical evidence. Benefits of this workflow over preexisting methods are its speed of sample preparation, efficiency of sample use (particularly for small samples), flexibility by allowing for storage of excess extracted nuclei, and economy of scale. Together, these features support preparation of large numbers of cancer samples in a relatively short period of time. Citation Format: Haochen Zhang, Elias-Ramzey Karnoub, Ronan Chaligné, Ignas Masilionis, Alvin Makohon-Moore, Jungeui Hong, Christine Iacobuzio-Donahue. Optimization of high-throughput, high-depth, targeted single-cell DNA sequencing to pancreatic ductal adenocarcinoma [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 59.