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Leveraging massively parallel reporter assays for evolutionary questions.

ABSTRACT: A long-standing goal of evolutionary biology is to decode how gene regulation contributes to organismal diversity. Doing so is challenging because it is hard to predict function from non-coding sequence and to perform molecular research with non-model taxa. Massively parallel reporter assays (MPRAs) enable the testing of thousands to millions of sequences for regulatory activity simultaneously. Here, we discuss the execution, advantages, and limitations of MPRAs, with a focus on evolutionary questions. We propose solutions for extending MPRAs to rare taxa and those with limited genomic resources, and we underscore MPRA's broad potential for driving genome-scale, functional studies across organisms.

SUBMITTER: Gallego Romero I

PROVIDER: S-EPMC9926830 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Leveraging massively parallel reporter assays for evolutionary questions.

Gallego Romero Irene I Lea Amanda J AJ

Genome biology 20230214 1

A long-standing goal of evolutionary biology is to decode how gene regulation contributes to organismal diversity. Doing so is challenging because it is hard to predict function from non-coding sequence and to perform molecular research with non-model taxa. Massively parallel reporter assays (MPRAs) enable the testing of thousands to millions of sequences for regulatory activity simultaneously. Here, we discuss the execution, advantages, and limitations of MPRAs, with a focus on evolutionary que ...[more]

PMID: 36788564

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Project description:MotivationThe majority of the human genome is composed of non-coding regions containing regulatory elements such as enhancers, which are crucial for controlling gene expression. Many variants associated with complex traits are in these regions, and may disrupt gene regulatory sequences. Consequently, it is important to not only identify true enhancers but also to test if a variant within an enhancer affects gene regulation. Recently, allele-specific analysis in high-throughput reporter assays, such as massively parallel reporter assays (MPRAs), have been used to functionally validate non-coding variants. However, we are still missing high-quality and robust data analysis tools for these datasets.ResultsWe have further developed our method for allele-specific analysis QuASAR (quantitative allele-specific analysis of reads) to analyze allele-specific signals in barcoded read counts data from MPRA. Using this approach, we can take into account the uncertainty on the original plasmid proportions, over-dispersion, and sequencing errors. The provided allelic skew estimate and its standard error also simplifies meta-analysis of replicate experiments. Additionally, we show that a beta-binomial distribution better models the variability present in the allelic imbalance of these synthetic reporters and results in a test that is statistically well calibrated under the null. Applying this approach to the MPRA data, we found 602 SNPs with significant (false discovery rate 10%) allele-specific regulatory function in LCLs. We also show that we can combine MPRA with QuASAR estimates to validate existing experimental and computational annotations of regulatory variants. Our study shows that with appropriate data analysis tools, we can improve the power to detect allelic effects in high-throughput reporter assays.Availability and implementationhttp://github.com/piquelab/QuASAR/tree/master/mpra.Contactfluca@wayne.edu or rpique@wayne.edu.Supplementary informationSupplementary data are available online at Bioinformatics.

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Leveraging massively parallel reporter assays for evolutionary questions.

Publications

Leveraging massively parallel reporter assays for evolutionary questions.

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