Clonal Analysis of Patient-Derived Samples Using Cellular Barcodes.

ABSTRACT

Cellular barcoding is a relatively simple method that allows quantitative assessment of the clonal dynamics of normal, nonmalignant hematopoietic stem cells and of leukemia. Cellular barcodes are (semi-)random synthetic DNA sequences of a fixed length, which are used to uniquely mark and track cells over time. A successful barcoding experiment consists of several essential steps, including library production, transfection, transduction, barcode retrieval, and barcode data analysis. Key challenges are to obtain sufficient number of barcoded cells to conduct experiments and reliable barcode data analysis. This is especially relevant for experiments using primary leukemia cells (which are of limited availability and difficult to transduce), when studying low levels of chimerism, or when the barcoded cell population is sorted in different smaller subpopulations (e.g., lineage contribution of normal hematopoietic stem cells in murine xenografts). In these settings, retrieving accurate barcode data from low input material using standard PCR amplification techniques might be challenging and more sophisticated approaches are required. In this chapter we describe the procedures to transfect and transduce patient-derived leukemia cells, to retrieve barcoded data from both high and low input material, and to filter barcode data from sequencing noise prior to quantitative clonal analysis.