Simple Copy Number Determination with Reference Query Pyrosequencing (RQPS)

Simple Copy Number Determination with Reference Query Pyrosequencing (RQPS)

Zhenyi Liu^1,3, Daniel L. Schneider¹, Kerry Kornfeld¹, and Raphael Kopan^{1,2,3 1} Department of Developmental Biology, School of Medicine, Washington University, St. Louis, Missouri 63110, USA
² Division of Dermatology, Department of Medicine, School of Medicine, Washington University, St. Louis, Missouri 63110, USA

The accurate measurement of the copy number (CN) for an allele is often desired. We have developed a simple pyrosequencing-based method, reference query pyrosequencing (RQPS), to determine the CN of any allele in any genome by taking advantage of the fact that pyrosequencing can accurately measure the molar ratio of DNA fragments in a mixture that differ by a single nucleotide. The method involves the preparation of an RQPS probe, which contains two linked DNA fragments that match a reference allele with a known CN and a query allele with an unknown CN.