A dinucleotide tag-based parallel reporter gene assay method

The causal SNPs leading to increased cancer predisposition mainly function as gene regulatory elements, the evaluation of which largely rely on the parallel reporter gene assay system. However, the common DNA barcode-based parallel reporter gene assay systems are troubled with tag bias, mainly due to the tag nucleotide composition. Here we describe a versatile dinucleotide-tag reporter system (DiR) that enables parallel analysis of regulatory elements with minimized bias based on the next-generation sequencing. The DiR system is also more robust than the classical luciferase assay method, especially in the investigation of moderate-level regulatory elements. We applied DiR-seq assay in the functional evaluation of the prostate cancer risk SNPs in prostate cancer cell lines and disclosed 2, and 6 regulatory SNPs in PC-3 and LNCaP cells. The DiR system has great potential to advance the functional study of risk SNPs that have associations with polygenic diseases.