EvaGreen-Based Droplet Digital PCR: A Rapid and Inexpensive Method to Confirm and Enhance Breakpoint Resolution of CNVs Detected by CMA

Chromosomal microarray (CMA) reliably detects copy number variations (CNVs). Depending on the probe coverage in a region of interest, however, smaller CNVs and the inclusion/exclusion of specific genes or exons near the CNV breakpoints may require confirmation by alternate methods. Hydrolysis probe-based qPCR has been widely utilized; however, it has several issues such as relative quantification, waiting time for the probe production, and probe costs. Here we report development of a rapid copy number (CN) confirmation assay using droplet digital PCR (ddPCR) with the dsDNA-binding fluorescent dye, EvaGreen. As a proof-of-concept, we tested samples with known 15q11.2 CNVs. Normalized CNs for gains and losses were 2.95 ± 0.06 (n = 6 in triplicate) and 0.98 ± 0.05 (n = 7, in triplicate), respectively, when normalized to RPPH1 (mean±SD). CN normalization to the TERT gene generated similar results. Compared to qPCR, ddPCR demonstrated superior results and workflow. Subsequently, we applied this ddPCR technique to a clinical sample with a single CN loss in the autosomal dominant AUTS2 gene. This loss clearly involved a large intronic region but the inclusion of several neighboring exons identified by analysis software was unclear upon manual review due to sparse probe coverage. In less than 48 hours we were able to design, order, and receive primers, and perform ddPCR testing that excluded loss of the exons in question. This prevented a potentially false-positive, pathogenic CNV call. We conclude that EvaGreen-based ddPCR is a suitable method for rapid and robust confirmation of CNV results identified by CMA.