Probing Copy Number Variations Using Bio-Rad’s QX100 Droplet Digital PCR System

Need for Improved Methods to Validate, Diagnose, and Monitor Copy Number Variations Given the high incidence and clinical impact of CNVs, a precise, rapid, and cost-effective method is needed for high-throughput validation of candidate CNV associations and for subsequent routine deployment in diagnostic settings. The predominant method used to validate CNVs in larger populations is real-time or quantitative PCR (qPCR), which measures the relative rates of fluorescence increases during the exponential amplification of target and singlecopy reference genes. The accuracy and precision of these measurements can be impacted by multiple factors including differences in amplification rates between the target and reference genes, variations in their amplification rates during qPCR, sampling error due to DNA concentration, and analysis errors (Karlen et al. 2007). Weaver et al. (2010) rigorously characterized these factors and found that systemic errors can be addressed by increasing the number of replicates to achieve the desired precision. However, the required number of replicates increases rapidly as finer discrimination is desired, with four replicates required to distinguish a twofold difference (for example, a CNV of 1 vs. 2) and up to 18 replicates to distinguish a 1.25-fold difference (for example, a CNV of 4 vs. 5).