A tool for the design of high-throughput massively parallel reporter assays (MPRAs)

DNA synthesis and sequencing technology is advancing rapidly, allowing for the design of high-throughput experiments which were previously hindered by technological constraints. Massively Parallel Reporter Assay experiments (MPRAs) is a novel method, that uses the technological advances and the reduction in the associated cost, to study the regulatory activities of tens or hundreds of thousands of DNA oligonucleotides. The oligonucleotides are synthesized using microarrays, each of which contains a uniquely identifiable barcode. Next, the oligonucleotides are amplified, put into vectors with a reporter gene and transfected into cells. By measuring the expression levels of the reporter gene, the regulatory properties of the corresponding sequence can be inferred. Therefore, MPRAs and other similar high-throughput methods, enable the systematic examination of regulatory synthetic sequences to identify rules that govern regulation of gene expression or to examine the type of regulatory role of specific elements, or motif positioning.

For instance, some questions that can be explored using synthetic sequences are:

  • The regulatory effect of the positioning of single or combinations of Transcription Factor Binding Sites (TFBSs) such as their relative distance from one another, or their distance from transcription-start sites.
  • The type of regulatory effect of a specific element i.e. repressor or activator and the magnitude of its regulatory effect.
  • The role of homotypic and heterotypic clusters within regulatory elements and potential synergistic effects.
  • The influence of the sequence context and flanking sites at the vicinity TFBSs.

Additionally, MPRAs allow for the effect of regulatory Single Nucleotide Polymorphisms, small insertions or small deletions regarding gene expression. By designing oligonucleotide sequences that differ specifically at a SNP, or contain a small insertions or a small deletions the regulatory role of variants found in the human genome can be examined.

Overall, MPRAs and associated technologies are very powerful, but currently systematically designing these type of experiments remains very challenging. We provide MPRAnator, a novel tool for the design of MPRA experiments to study motifs or polymorphisms in a systematic manner.

The first module allows the user to place different combinations of transcription factor binding motifs.

The second module allows the user to explore different combinations of genetic variants from a VCF file.

The third module allows for the synthesis of various controls for the experiments.

In the first two cases we allow for a a modular design whereby spacers, restriction sites and barcodes can be arranged as desired.

MPRAnator tools can be accessed programmatically by modifying the following example codes:

MPRAnator Motif:

wget -O- ';reverseComplement=yes;minSpacing=2;maxSpacing=4;leftDistance=3;rightDistance=3;frequencyOfInsertion=5;barCodeLength=12;minimumGCContent=20;maximumGCContent=80;barCodeDistance=2;numOfBarCodesPerSequence=4;restriction1=;restriction2=;adapter1=;adapter2=;ordering=bg,bc=;sequencesS=%3Esequence1%0AAGAGATTAGTCAGTACGGCTAGCTAGCTACGTCTATATTATAGCGATACGGG;motifS=%3Emotif1%0AGGCTT%0Amotif2%0AAAACGG' 2>/dev/null

MPRAnator SNP:

wget -O- ';makeSnpCombinations;barCodeLength=12;minimumGCContent=20;maximumGCContent=80;barCodeDistance=2;numOfBarCodesPerSequence=4;restriction1=;restriction2=;adapter1=;adapter2=;ordering=bg,bc=;sequencesS=>sequence1chr1:42990-43041,testest%0AACAGATTAGTCAGTACGGCTAGCTAGCTACGTCTATATTATAGCGATACGGG;SnpS=1%0942991%09rs43434%09T%09G%0A1%0943030%09rs121212%09A%09G' 2>/dev/null

MPRAnator Transmutation:

wget -O- ';seq_string=>FRED%0AACAACAGAGAGACATCATCGACGATGCTGA' 2>/dev/null

MPRAnator is also available at:

The full description of MPRAnator is at:

Sanger Institute Contributors

Previous contributors

Photo of Dr Martin Hemberg

Dr Martin Hemberg

Former CDF Group Leader

External Contributors

Photo of Naman Jain

Naman Jain