Abstract
Motivation: Genomic copy number variation (CNV) can influence susceptibility to common diseases. High-throughput measurement of gene copy number on large numbers of samples is a challenging, yet critical, stage in confirming observations from sequencing or array Comparative Genome Hybridization (CGH). The paralogue ratio test (PRT) is a simple, cost-effective method of accurately determining copy number by quantifying the amplification ratio between a target and reference amplicon. PRT has been successfully applied to several studies analyzing common CNV. However, its use has not been widespread because of difficulties in assay design.Results: We present PRTPrimer (www.prtprimer.org) software for automated PRT assay design. In addition to stand-alone software, the web site includes a database of pre-designed assays for the human genome at an average spacing of 6 kb and a web interface for custom assay design. Other reference genomes can also be analyzed through local installation of the software. The usefulness of PRTPrimer was tested within known CNV, and showed reproducible quantification. This software and database provide assays that can rapidly genotype CNV, cost-effectively, on a large number of samples and will enable the widespread adoption of PRT.Availability: PRTPrimer is available in two forms: a Perl script (version 5.14 and higher) that can be run from the command line on Linux systems and as a service on the PRTPrimer web site (www.prtprimer.org).Contact: cjt14@le.ac.ukSupplementary Information: Supplementary data are available at Bioinformatics online.
Highlights
Copy number variation (CNV) is a pervasive and extensive source of variation between individual genomes in humans and many other species
(2) Indication whether amplicons lay within an annotated copy number variation (CNV) or indel [annotation according to the database of genomic variants (DGV)]
We describe a resource of off-the-shelf paralogue ratio test (PRT) assays as well as for custom design of assays
Summary
Copy number variation (CNV) is a pervasive and extensive source of variation between individual genomes in humans and many other species. The multiplex ligation-dependent amplification method is used routinely in clinical genetics for screening disease genes for exons, and has been shown to accurately infer integer copy number even on challenging multicopy loci (Eijk-Van Os and Schouten, 2011) It is propriety, expensive and commercially available only for certain loci, and often 100–200 ng of DNA is required. PRT is a form of quantitative PCR that differs from the conventional method in one important aspect: by careful design of the primers targeting paralogous sequences, one primer pair is used to amplify a putative CNV target locus relative to a single copy reference locus (Armour et al, 2007) This makes the kinetics of target and reference amplification similar, and results in increased accuracy of integer copy number calling. The software can be installed locally or run through the web resource, is optimized for multicore systems and can be adapted to use genomes from other species
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