Best practices for perturbation MPRA-a computational evaluation framework of sequence design strategies

The advent of the perturbation-based massively parallel reporter assays (MPRAs) technique has enabled delineating of the roles of non-coding regulatory elements in orchestrating gene expression. However, computational efforts remain scant to evaluate and establish guidelines for sequence design strategies for perturbation MPRAs. Here, we propose a framework for evaluating and comparing various perturbation strategies for MPRA experiments. Under this framework, we benchmark three different perturbation approaches from the perspectives of alteration in motif-based profiles, consistency of MPRA outputs, and robustness of models that predict the activities of putative regulatory motifs. Although our analyses show similar while significant results in multiple metrics, the method of randomly shuffling nucleotides outperform the other two methods. Thus, we still recommend designing sequences by randomly shuffling the nucleotides of the perturbed site in perturbation-MPRA. The evaluation framework, together with the benchmarking findings in our work, creates a resource of computational pipelines and illustrates the promise of perturbation-MPRA for predicting non-coding regulatory activities.