Combining cell sorting and ResolveDNA single-cell genomic amplification for exposing intratumoral heterogeneity

A comprehensive picture of somatic cell clonal evolution driving tumorigenesis is not possible with bulk sequencing strategies that fail to uncover rare alleles. Single-cell analysis provides the fundamental unit of resolution to define this evolution, but existing methods to amplify the genomes of single cells suffer from poor genomic coverage, uniformity, and allelic balance.

In this SelectScience webinar, now available on demand, Dr. Jon Zawistowski, senior director of R&D at BioSkryb Genomics, discusses how cells enriched from heterogeneous tumors by cell sorting can be used upstream of the ResolveDNA™ chemistry to concomitantly improve the sensitivity of variant allele detection.

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For what other applications have you used immunoenrichment upstream of your genome amplification?

JZ: In addition to our EpCAM work where we've been utilizing immunoenrichment for breast cancer, we can also iterate upon these experiments and define new markers both in the context of primary cells as well as cell line models. One example we have is where we've been looking at primary airway epithelial cells. Within this heterogeneous population of airway cells, there are basal stem cells. One of the applications is to isolate these stem cells by immunoenrichment. In this application, we're utilizing a neuronal growth factor receptor and then employing that antibody using upstream fluorescence-activated cell sorting (FACS), and then proceeding with primary template-directed amplification (PTA). This will allow us to gain insight into the biology of these airway epithelial cells and how different environmental exposures contribute to genomic variation in that system.

How many colors have you successfully employed for coupling PTA and FACS with the Sony SH800 Cell Sorter?

JZ: We currently have a collaboration looking at leukemia, and with the wealth of knowledge about cell surface expression, we're able to successfully move up to six colors with the existing laser configuration on the Sony SH800 Cell Sorter. We have four additional colors/antibodies that we use to monitor the cell surface expression in these primary leukemia samples. This has implications for immunotherapy and other clinical implications going forward, in addition to our primary antibodies that we use for sorting.

Have you sorted nuclei for PTA using the Sony SH800 instrument?

JZ: Throughout this talk, I gave examples of singulated primary cells where we've sorted directly into wells and done the amplification chemistry on intact single cells. We realize, especially in the context of neurology, that nuclei are the desired way to move forward.

We have a nice study in primary meningeal tissue, where we were able to singulate the cells from primary tissue, and then take those singulated cells and use the Sony SH800 Cell Sorter. In that case, we were confirming the presence of nuclei versus cells using a propidium iodide strategy. Using the sorter and the propidium iodide strategy in upstream singulation, we were able to obtain excellent genomic amplification of these single nuclei.

To learn more about ResolveDNA^TM single-cell genomic amplification, watch the full webinar here>>

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