Single nucleus DNA sequencing of flow sorted archived frozen and formalin fixed paraffin embedded solid tumors

Background: Archived samples, including frozen and formalin fixed paraffin embedded (FFPE) tissues, are a vast resource of clinically annotated materials for the application of high-definition genomics to improve patient management and provide a molecular basis for the delivery of personalized cancer therapeutics. Notably, FFPE tissues are stable, provide repeat sampling of tissues of interest, and can be stored indefinitely at ambient temperature. The development of single cell DNA sequencing (scDNA-seq) technologies provides an unparalleled opportunity for the study of tumor heterogeneity and the identification of often rare subclonal cell populations that drive tumor evolution and progression to advanced therapy resistant disease. However, major limitations to the use of archived tissues for scDNA-seq include the low yields of intact cells in the presence of high levels of subcellular debris in biopsies, and the highly variable quantity and quality of the DNA extracted from samples of interest. The latter is of high significance for the use of FFPE tissues due to the presence of DNA-protein crosslinks. In addition, many samples, notably tumors arising in solid tissues, contain admixtures of reactive stroma, inflammatory cells, and necrosis in immediate contact with tumor cells.

Results: To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived fresh frozen (FF) and FFPE tumor tissues. Our methods, which include isolation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching FF and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest.

Conclusions: Our flow sorting based protocol provides intact nuclei suitable for snDNA-seq from archival FF and FFPE tissues. Furthermore, we have developed QC steps that optimize the preparation and selection of samples for deep single cell clonal profiling. Our data processing pipeline captures rare subclones in tumors with highly variable genomes based on variants in genes of interest.