A systematic evaluation of the design, orientation, and sequence context dependencies of massively parallel reporter assays

Enhancers play important roles in evolution and disease. However, traditional assays to test enhancers are low throughput and not scalable to the >100,000 enhancers in the human genome. To better prioritize variants associated with disease and to study the role of enhancers, our group and others developed massively parallel reporter assays (MPRAs), which functionally screen thousands of sequences for regulatory activity in parallel. Although MPRAs have been applied to address diverse questions in gene regulation, there has been no systematic comparison of how differences in experimental design influence findings, making it difficult to interpret results and compare between groups. Here, we screen a library of 2,440 sequences, representing candidate liver enhancers and controls, in HepG2 cells for regulatory activity using nine different approaches (including conventional episomal, STARR-seq, and lentiviral MPRA designs). We identify subtle but significant differences in the resulting measurements that correlate with epigenetic and sequence-level features. We also test this library in both orientations with respect to the promoter, validating en masse that enhancer activity is robustly independent of orientation. Finally, we develop and apply a novel method to assemble and functionally test libraries of the same putative enhancers as 192-mers, 354-mers, and 678-mers, and observe surprisingly large differences in functional activity. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results. We systematically cloned the same library of 2,440 candidate enhancers into nine different MPRA designs. These included episomal vs lentivirally integrated constructs as well as STARR-seq and traditional upstream reporter assays. We also synthesized a subset of the 2,440 candidate sequences as 192bp, 354bp, and 678bp sequences to test for the effect of added sequence context on activity. Finally, we directionally cloned the full library in both directions to compare effects of direction on activity. All libraries were transfected into the human liver hepatocellular carcinoma cell line, HepG2.