Figure 2.

Principle of massively parallel reporter assays (MPRAs). MPRAs are used to simultaneously test hundreds to thousands of variants for potential regulatory effects in one assay. (A) Generation of MPRA pools. First, the genomic sequence around each variant (candidate regulatory elements [CREs]) is synthesized or otherwise derived and then combined with an individual barcode. All barcoded sequences are combined into one pool. (B) MPRA reporter assay. This pool is then cloned into vectors. The vectors contain a reporter gene (potentially driven by a minimal promoter) and place the CRE upstream of the transcriptional start site, while the barcode becomes part of the transcript’s 3′ or 5′ untranslated region. Depending on the assay type, the vectors are designed to either remain episomal or integrate into the genome (e. g., by lentivirus). The vector pool is then transfected (or transduced) into cell types of interest where the reporter genes are expressed. Following extraction of DNA and RNA from those cells, barcodes can be converted into highly complex sequencing libraries and read out on a high-throughput sequencing device. A regulatory effect of a certain CRE can be inferred from the number of detected barcode sequences at the RNA level, corrected by the number of transfected plasmids (detected by the barcode abundance in DNA). Allelic effects are derived from comparing the inferred expression effect of CREs with and without the allele of interest.