Fig. 1.

Experimental design. (A) We synthesized 137-bp MPRA fragments overlapping 32,776 hSubs in 3,027 HGEs and 1,363 HARs. (B) MPRA fragments (human in blue, chimpanzee in green) were cloned in front of a luc2 reporter gene and a random oligonucleotide barcode tag (in yellow). Sequencing and counting barcodes provide a quantitative measure of enhancer activity. (C) In stage 1 of the experiment, we screened 50,268 orthologous human–chimpanzee fragment pairs. In stage 2, the impact of genetic differences within all fragments exhibiting species-specific changes in activity was dissected by testing all possible combinations of hSubs and ancestral states in both the human and chimpanzee background reference sequences.