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

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

PROVIDER: PRJNA598033 | ENA |

REPOSITORIES: ENA

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			Action	DRS
		Other
	SRR10800981_1.fastq.gz	Fastqsanger.gz
	SRR10800981_2.fastq.gz	Fastqsanger.gz
	SRR10800982_1.fastq.gz	Fastqsanger.gz
	SRR10800982_2.fastq.gz	Fastqsanger.gz

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

Project description: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.

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Project description:This GEO submission accompanies the study “Best Practices for Massively Parallel Reporter Assays – Analyses, Diagnostics, and Interpretation” and includes five MPRA datasets used as illustrative examples in the manuscript figures. The datasets represent common MPRA experimental designs and are provided to demonstrate recommended analytical workflows, quality-control diagnostics, and interpretation strategies. These data serve as reference resources for analysis benchmarking, rather than for deriving new biological conclusions.

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