Jason C. Klein, Vikram Agarwal, Fumitaka Inoue, Aidan Keith, Beth Martin et al.
Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. To date, there has been no systematic comparison of differences in MPRA design. Here, we screen a library of 2,440 candidate liver enhancers and controls for regulatory activity in HepG2 cells using nine different MPRA designs. We identify subtle but significant differences that correlate with epigenetic and sequence-level features, as well as differences in dynamic range and reproducibility. We also validate en masse that enhancer activity is robustly independent of orientation, at least for our library and designs. Finally, with a new method, we assemble and test the same enhancers as 192-mers, 354-mers, and 678-mers, and observe surprisingly large differences. 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.
Jason C. Klein, Vikram Agarwal, Fumitaka Inoue, Aidan Keith, Beth Martin, Martin Kircher, Nadav Ahituv, Jay Shendure. A systematic evaluation of the design and context dependencies of massively parallel reporter assays. 2020-10-12. https://doi.org/10.1038/s41592-020-0965-y
The common 2,440-element library was packaged with wild-type lentiviral integrase, placing 171-bp candidate enhancers upstream of a minimal promoter and linked barcodes in the reporter 3' UTR. Three independent HepG2 infections were profiled by matched DNA and RNA barcode sequencing.
The common 2,440-element library was inserted into the original human STARR-seq (HSS) reporter, placing enhancer sequences in the reporter 3' UTR with adjacent linked barcodes. The episomal library was transfected into HepG2 cells in three independent replicate cultures.
A 2,336-element 192-bp enhancer/control library was directionally cloned in both orientations upstream of the pGL4.23c minimal promoter and transfected into HepG2 cells. Four independent replicate cultures were profiled by matched DNA and RNA barcode sequencing.
The same candidate liver-enhancer library was assembled as short (192-bp), medium (354-bp), and long (678-bp) genomic-context sequences, cloned into pGL4.23c, pooled, and transfected into HepG2 cells. Four independent replicate cultures were profiled by matched DNA and RNA barcode sequencing.
The common 2,440-element library was packaged with mutant lentiviral integrase, leaving 171-bp candidate enhancers upstream of a minimal promoter with linked barcodes in the reporter 3' UTR as nonintegrated/episomal reporter DNA. Three independent HepG2 infections were profiled by matched DNA and RNA barcode sequencing.
The common 2,440-element library was packaged with wild-type lentiviral integrase, placing 171-bp candidate enhancers upstream of a minimal promoter and linked barcodes in the reporter 5' UTR. Three independent HepG2 infections were profiled by matched DNA and RNA barcode sequencing.
The common 2,440-element library was packaged with mutant lentiviral integrase, leaving the 171-bp enhancer/minimal-promoter reporter library nonintegrated/episomal with linked barcodes in the reporter 5' UTR. Three independent HepG2 infections were profiled by matched DNA and RNA barcode sequencing.
The common 2,440-element library was packaged with mutant lentiviral integrase, leaving the enhancer-plus-barcode reporter library nonintegrated/episomal in HepG2 cells. Three independent infections were profiled by matched DNA and RNA barcode sequencing.
The common 2,440-element library was cloned with 171-bp candidate liver enhancers upstream of the pGL4.23c minimal promoter and reporter, with linked barcodes in the reporter 3' UTR. Plasmid libraries were transfected into HepG2 cells in three independent replicate cultures.
The common 2,440-element library was tested in the ORI promoterless STARR-seq design, with enhancer sequences and adjacent barcodes in the reporter 3' UTR. The episomal library was transfected into HepG2 cells in three independent replicate cultures.
The common 2,440-element library was packaged with wild-type lentiviral integrase, placing both the 171-bp candidate enhancer and its linked barcode in the reporter 3' UTR. Three independent HepG2 infections were profiled by matched DNA and RNA barcode sequencing.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 24 files (ZIP)GSE142696_9MPRA.ActivityRatios.IndividualReps.tsv.gzGSE142696_9MPRA.ActivityRatios.tsv.gzGSE142696_family.soft.gzGSE142696_ForwardReverse.ActivityRatios.IndividualReps.tsv.gzGSE142696_ForwardReverse.ActivityRatios.tsv.gzGSE142696_ForwardReverse_sequences_coordinates.tsv.gzGSE142696_RAW/GSM4237951_shortMedLong_assignments.tsv.gzGSE142696_RAW/GSM4237951_shortMedLong_elements.fa.gzGSE142696_RAW/GSM4237951_ShortMedLong_sequences_coordinates.tsv.gzGSE142696_RAW/GSM4237952_fwd_assignments.tsv.gzGSE142696_RAW/GSM4237952_fwd_elements.fa.gzGSE142696_RAW/GSM4237953_rev_assignments.tsv.gzGSE142696_RAW/GSM4237953_rev_elements.fa.gzGSE142696_RAW/GSM4237954_9MPRA_assignments.tsv.gzGSE142696_RAW/GSM4237954_9MPRA_elements.fa.gzGSE142696_RAW/GSM4237954_9MPRA_sequences_coordinates.tsv.gzGSE142696_RAW.tarGSE142696_ShortMedLong.ActivityRatios.IndividualReps.tsv.gzGSE142696_ShortMedLong.ActivityRatios.tsv.gzREADME.txtSupplementary_Table_1.xlsxSupplementary_Table_2.xlsxSupplementary_Table_6.xlsxSupplementary_Table_7.xlsx