High-throughput Single-cell Targeted DNA Sequencing from Tumor and Metastatic Samples Reveal Spatial Resolution of Evolutionary Trajectory Routes to Clonal Propagation

Abstract
Recent advancements in genomic analysis of tumors have revealed that cancer disease evolves by a reiterative process of somatic variation, clonal expansion and selection. Therefore, intra- and inter-tumor genomic heterogeneity has become a major area of investigation. While bulk NGS has contributed significantly to our understanding of cancer biology and genomics, the genetic heterogeneity of a tumor at the individual cellular level is masked with the average readout provided by a bulk measurement. Very high bulk sequence read depths are required to identify lower prevalence mutations. Even at these high read depths, confidence confirmation in events at the 1% range or less is a formidable challenge. Rare events and mutation co-occurrence within and across select population of cells are obscured with such average signals. Additionally, recent reports highlight some of the crucial issues in whole exome studies for false detection rates. In an effort to explore this biology at higher resolution, we conducted single-cell targeted DNA analysis with the Mission Bio Tapestri™ Platform using sectioned melanoma metastatic tissues. Leveraging proprietary droplet microfluidics, the workflow unlocks access to gDNA, enabling high coverage uniformity of ~90% and low ADO of ~10%. Up to 20,000 cells can be interrogated with catalog or custom amplicon panels for any solid tumor type. Here, we use the Tapestri Single-Cell DNA Tumor Hotspot Panel that targets 59 commonly-mutated genes across 244 amplicons. We report that an analysis of multiple spatially-separated samples obtained from distinct metastatic sites in subjects revealed unique genomic signatures mapping to each solitary sample. These datasets support a number of conclusions including:
1) our recently optimized universal nuclei extraction process removes cellular components that are known to be highly inhibitory to PCR amplification – in this case melanin from melanoma cells,
2) single-cell analysis correlates strongly with bulk sample analysis, enabling confident comparison with previously-acquired results,
3) rare subclones, present at 0.15%, were detected, which is critical when monitoring disease progression,
4) single-cell analysis unambiguously identified the clones in each sample, enabling the reconstruction of clonal phylogeny,
5) the different clonal lineage observed in distant metastatic tissues highlights complex disease progression, and
6) tumor purity was measured at the single-cell genetic level. In summary, single-cell analysis offers to overcome the limitations of bulk NGS and can provide unique insights into the cellular-level complexities of tumor heterogeneity and phylogenesis.
Here, the use of the Tapestri Platform demonstrates the power of single-cell DNA sequencing for characterizing solid tumor tissue samples and understanding disease evolution.