Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. To date, there are limited studies that systematically compare differences in MPRA design. Here, we screen a library of 2,440 candidate liver enhancers and controls for regulatory activity in HepG2 cells using nine different MPRA designs. We identify subtle but significant differences that correlate with epigenetic and sequence-level features, as well as differences in dynamic range and reproducibility. We also validate that enhancer activity is largely independent of orientation, at least for our library and designs. Finally, we assemble and test the same enhancers as 192-mers, 354-mers and 678-mers and observe sizable differences. 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.

大规模并行报告基因检测 (MPRA) 可并行筛选数千个序列的调控活性。迄今为止，系统比较 MPRA 设计差异的研究还很有限。在这里，我们使用九种不同的 MPRA 设计筛选了包含 2,440 种候选肝脏增强剂和 HepG2 细胞调节活性对照的库。我们发现了与表观遗传和序列水平特征相关的微妙但显着的差异，以及动态范围和再现性的差异。我们还验证了增强子活性在很大程度上与方向无关，至少对于我们的库和设计而言是如此。最后，我们组装并测试了与 192-mers、354-mers 和 678-mers 相同的增强子，并观察到相当大的差异。这项工作为高通量报告基因检测的实验设计提供了一个框架，表明被测元件的扩展序列背景以及在较小程度上的精确检测会影响 MPRA 结果。