Abstract 2532: High-throughput single-cell targeted DNA sequencing from frozen, fixed and preserved solid tumor samples reveals complex genomic variation and clonal propagation
Authors: Ruff David , Mendez Pedro , Mendoza Daniel , Li Nianzhen , Sciambi Adam , Gokhale Kaustubh , Dhingra Dalia , Jones Keith , Eastburn Dennis 
Published: August 2019

Biologically annotated specimens such as frozen, fixed and preserved tissues are key sources of cells for genomic analysis. Bulk NGS using archived solid tumor samples is inadequate to fully characterize somatic variation buried in the landscape of cellular populations. Single-cell targeted DNA sequencing provides an essential solution to elucidate and map genomic variation in such materials. Although the study of frozen, fixed and preserved tissues at the single-cell level is compromised by preservation processes, the isolation of nuclei allows the recovery of suitable gDNA templates. Common tissue disaggregation processes can be complicated by persistence of conglomerated cellular components, ruptured nuclei, and other insoluble extracellular matrices. For challenging samples, we developed a nuclei isolation protocol that demonstrates optimal performance for high-quality targeted DNA sequencing from archived human solid tumor samples. This process begins with physical maceration of ~10-100 mg preserved tissue or 20-100 uM sections, suspension followed by enzymatic treatment, filtration and centrifugal collection. After cell straining, nuclei are ready for counting, staining and sequencing. Fluorescent microscopy using membrane, cytoplasmic and nuclear stains reveal highly purified intact nuclei, recovering at least 500K nuclei from 30-50 mg of tissue containing greater than 70% nucleated cells by H&E-staining. Many human samples provide outstanding quality nuclei suspensions, including cryopreserved cell lines, PBMCs, bone marrow, liver, brain, breast, colon, lung, prostate and melanomas. Fixed and preserved specimens also yield nuclei readily interrogated by targeted DNA sequencing panels. Nuclei suspensions are readily processed with the Tapestri Platform for single-cell DNA analysis. Leveraging droplet microfluidics and barcoding, the workflow enables high uniformity of ~90%, low ADO of ~10%, and typical nuclei recovery rates exceed 10%. Up to 20,000 nuclei can be interrogated in each run with catalog or custom panels for any tumor type. Here, a 59-gene tumor hotspot panel was used to study five melanoma metastases. The single-cell data enabled the unique reconstruction of tumor sample clonal phylogeny unresolved by bulk analysis. Also, low prevalence metastatic subclones masked in bulk NGS data were detected in normal liver samples. In summary, we show that diverse types of archived tumor tissues are readily dissociated with this universal nuclei protocol. Researchers now have a highly sensitive, targeted, customizable solution for revealing genomic variation and clonal propagation in complex archived solid tumor samples.

Citation Format: David Ruff, Pedro Mendez, Daniel Mendoza, Nianzhen Li, Adam Sciambi, Kaustubh Gokhale, Dalia Dhingra, Keith Jones, Dennis Eastburn. High-throughput single-cell targeted DNA sequencing from frozen, fixed and preserved solid tumor samples reveals complex genomic variation and clonal propagation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2532.