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

Klein, Jason C.; Agarwal, Vikram; Inoue, Fumitaka; Keith, Aidan M.; Martin, Beth; Kircher, Martin; Ahituv, Nadav; Shendure, Jay

doi:10.1101/576405

Cited by 15 publications

(27 citation statements)

References 42 publications

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“…We performed 5 technical replicates, including each allele in 5 oligos with different barcodes (10 barcodes per SNP). To maximize the number of assayed SNPs, each allele was only profiled in one direction, since concordance between the two directions has been shown to be high (Klein et al, 2019).…”

Section: Mpra Design and Methodsmentioning

confidence: 99%

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A screen of 1,049 schizophrenia and 30 Alzheimer’s-associated variants for regulatory potential

Myint¹,

Wang²,

Boukas³

et al. 2018

Preprint

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Recent genome-wide association studies (GWAS) identified numerous schizophrenia (SZ) and Alzheimer's disease (AD) associated loci, most outside protein-coding regions and hypothesized to affect gene transcription. We used a massively parallel reporter assay (MPRA) to screen, 1,049 SZ and 30 AD variants in 64 and 9 loci respectively for allele differences in driving reporter gene expression. A library of synthetic oligonucleotides assaying each allele 5 times was transfected into K562 chronic myelogenous leukemia lymphoblasts and SK-SY5Y human neuroblastoma cells. 148 variants showed allelic differences in K562 and 53 in SK-SY5Y cells, on average 2.6 variants per locus. Nine showed significant differences in both lines, a modest overlap reflecting different regulatory landscapes of these lines that also differ significantly in chromatin marks.Eight of nine were in the same direction. We observe no preference for risk alleles to increase or decrease expression. We find a positive correlation between the number of SNPs in Linkage Disequilibrium (LD) and the proportion of functional SNPs supporting combinatorial effects that may lead to haplotype selection. Our results prioritize future functional follow up of disease associated SNPs to determine the driver GWAS variant(s), at each locus and enhance our understanding of gene regulation dynamics.

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Section: Mpra Design and Methodsmentioning

confidence: 99%

“…For each SNP we include 5 oligos (technical replicates) for each allele. To increase the number of assayed SNPs, each allele was only profiled in one direction as it has been shown that there is high concordance between the two directions (Klein et al, 2019). We synthesized a 95 bp region surrounding each allele.…”

Section: Mpra Design and Quality Controlsmentioning

confidence: 99%

A screen of 1,049 schizophrenia and 30 Alzheimer’s-associated variants for regulatory potential

Myint¹,

Wang²,

Boukas³

et al. 2018

Preprint

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“…Numerous high-throughput sequencing methods identify enhancers using either plasmid or integrated reporter constructs and are collectively known as massively parallel reporter assays (MPRAs). While these assays offer unprecedented throughput for surveying genome function, their technical biases and limitations are a focus of ongoing research and optimization [23][24][25] . For example, most published MPRAs have been limited to short synthetic sequences (50-150 bp), despite the precise size of genomic enhancers remaining unknown 11 .…”

Section: Introductionmentioning

confidence: 99%

Transcription imparts architecture, function, and logic to enhancer units

Tippens

Liang

Leung

et al. 2019

Preprint

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Distal enhancers remain one of the least understood regulatory elements with pivotal roles in development and disease. We used massively parallel reporter assays to perform functional comparisons of two leading enhancer models and find that gene-distal transcription start sites (TSSs) are robust predictors of enhancer activity with higher resolution and specificity than histone modifications. We show that active enhancer units are precisely delineated by active TSSs, validate that these boundaries are sufficient to capture enhancer function, and confirm that core promoter sequences are required for this activity. Finally, we assay pairs of adjacent units and find that their cumulative activity is best predicted by the strongest unit within the pair.Synthetic fusions of enhancer units demonstrate that adjacency imposes winner-takes-all logic, revealing a simple design for a maximum-activity filter of enhancer unit outputs. Together, our results define fundamental enhancer units and a principle of non-cooperativity between adjacent units.

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“…While the RET SNPs were several hundred kilobases apart, another challenge is with variants in very close proximity, potentially within the same regulatory element. While oligo synthesis is limited in length, MPRA-based assay of such variants spanning up to 700bp is now possible with use of PCR amplification of oligos with uniquely complimentary ends (93). Likewise, 'capture-and-clone' MPRA designs, often using STARR-seq architecture to simplify cloning, fragment genomic DNA or capture e.g.…”

Section: The Utility Of Mpras For Parsing Linked Variationmentioning

confidence: 99%

The Oft-Overlooked Massively Parallel Reporter Assay: Where, When, and Which Psychiatric Genetic Variants are Functional?

Mulvey¹,

Lagunas²,

Dougherty³

2020

Preprint

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Neuropsychiatric phenotypes have been long known to be influenced by heritable risk factors. The past decade of genetic studies have confirmed this directly, revealing specific common and rare genetic variants enriched in disease cohorts. However, the early hope for these studies-that only a small set of genes would be responsible for a given disorder-proved false. The picture that has emerged is far more complex: a given disorder may be influenced by myriad coding and noncoding variants of small effect size, and/or by rare but severe variants of large effect size, many de novo.Noncoding genomic sequences harbor a large portion of these variants, the molecular functions of which cannot usually be inferred from sequence alone. This creates a substantial barrier to understanding the higher-order molecular and biological systems underlying disease risk. Fortunately, a proliferation of genetic technologies-namely, scalable oligonucleotide synthesis, high-throughput RNA sequencing, CRISPR, and CRISPR derivatives-have opened novel avenues to experimentally identify biologically significant variants en masse. These advances have yielded an especially versatile technique adaptable to large-scale functional assays of variation in both untranscribed and untranslated regulatory features: Massively Parallel Reporter Assays (MPRAs). MPRAs are powerful molecular genetic tools that can be used to screen tens of thousands of predefined sequences for functional effects in a single experiment. This approach has several ideal features for psychiatric genetics, but remains underutilized in the field to date. To emphasize the opportunities MPRA holds for dissecting psychiatric polygenicity, we review here its applications in the literature, discuss its ability to test several biological variables implicated in psychiatric disorders, illustrate this flexibility with a proof-of-principle, in vivo cell-type specific implementation of the assay, and envision future outcomes of applying MPRA to both computational and experimental neurogenetics.

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

Cited by 15 publications

References 42 publications

A screen of 1,049 schizophrenia and 30 Alzheimer’s-associated variants for regulatory potential

A screen of 1,049 schizophrenia and 30 Alzheimer’s-associated variants for regulatory potential

Transcription imparts architecture, function, and logic to enhancer units

The Oft-Overlooked Massively Parallel Reporter Assay: Where, When, and Which Psychiatric Genetic Variants are Functional?

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