Analysis of Copy Number Variation Using the Paralogue Ratio Test (PRT)

Copy number variation (CNV), where a segment of DNA differs in copy number between different individuals, is an extensive and often underappreciated source of genetic variation within species. However, reliably determining copy number of a particular DNA sequence for a large number of samples can be challenging. Here, I describe and review the paralogue ratio test (PRT) in detail. PRT was developed to robustly type the CNV of the beta-defensin locus using small amounts of genomic DNA in a high-throughput manner, and has been applied successfully at many other loci. I discuss the strategies for designing successful PRT assays using both manual and bioinformatics methods, how to optimize experimental conditions, and approaches for analyzing the data. I discuss strengths and weaknesses of the approach, and how to troubleshoot results, as well as the range of problems to which PRT can be a potential solution.