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

Nat Methods. 2020 Nov;17(11):1083-1091. doi: 10.1038/s41592-020-0965-y. | PubMed

Jason C Klein^1,2, Vikram Agarwal^1,3, Fumitaka Inoue^4,5,6, Aidan Keith¹, Beth Martin¹, Martin Kircher^1,7,8, Nadav Ahituv^9,10, Jay Shendure^11,12,13

1. Department of Genome Sciences, University of Washington, Seattle, WA, USA.
2. Memorial Sloan Kettering Cancer Center, New York, NY, USA.
3. Calico Life Sciences LLC, South San Francisco, CA, USA.
4. Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA.
5. Institute for Human Genetics, University of California San Francisco, San Francisco, CA, USA.
6. Institute for the Advanced Study of Human Biology (WPI-ASHBi), Kyoto University, Kyoto, Japan.
7. Berlin Institute of Health (BIH), Berlin, Germany.
8. Charité - Universitätsmedizin Berlin, Berlin, Germany.
9. Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA. nadav.ahituv@ucsf.edu.
10. Institute for Human Genetics, University of California San Francisco, San Francisco, CA, USA. nadav.ahituv@ucsf.edu.
11. Department of Genome Sciences, University of Washington, Seattle, WA, USA. shendure@uw.edu.
12. Howard Hughes Medical Institute, Seattle, WA, USA. shendure@uw.edu.
13. Brotman Baty Institute for Precision Medicine, University of Washington, Seattle, WA, USA. shendure@uw.edu.

Abstract

Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. To date, there are limited studies that systematically compare 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 that enhancer activity is largely independent of orientation, at least for our library and designs. Finally, we assemble and test the same enhancers as 192-mers, 354-mers and 678-mers and observe sizable 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.