High-throughput characterization of genetic effects on DNA-protein binding and gene transcription

Many variants associated with complex traits are in noncoding regions and contribute to phenotypes by disrupting regulatory sequences. To characterize these variants, we developed a streamlined protocol for a high-throughput reporter assay, Biallelic Targeted STARR-seq (BiT-STARR-seq), that identifies allele-specific expression (ASE) while accounting for PCR duplicates through unique molecular identifiers. We tested 75,501 oligos (43,500 SNPs) and identified 2720 SNPs with significant ASE (FDR < 10%). To validate disruption of binding as one of the mechanisms underlying ASE, we developed a new high-throughput allele-specific binding assay for NFKB1. We identified 2684 SNPs with allele-specific binding (ASB) (FDR < 10%); 256 of these SNPs also had ASE (OR = 1.97, P-value = 0.0006). Of variants associated with complex traits, 1531 resulted in ASE, and 1662 showed ASB. For example, we characterized that the Crohn's disease risk variant for rs3810936 increases NFKB1 binding and results in altered gene expression.