Dustin Shigaki, Orit Adato, Aashish N. Adhikari, Shengcheng Dong, Alex Hawkins-Hooker et al.
The integrative analysis of high-throughput reporter assays, machine learning, and profiles of epigenomic chromatin state in a broad array of cells and tissues has the potential to significantly improve our understanding of noncoding regulatory element function and its contribution to human disease. Here, we report results from the CAGI 5 regulation saturation challenge where participants were asked to predict the impact of nucleotide substitution at every base pair within five disease-associated human enhancers and nine disease-associated promoters. A library of mutations covering all bases was generated by saturation mutagenesis and altered activity was assessed in a massively parallel reporter assay (MPRA) in relevant cell lines. Reporter expression was measured relative to plasmid DNA to determine the impact of variants. The challenge was to predict the functional effects of variants on reporter expression. Comparative analysis of the full range of submitted prediction results identifies the most successful models of transcription factor binding sites, machine learning algorithms, and ways to choose among or incorporate diverse datatypes and cell-types for training computational models. These results have the potential to improve the design of future studies on more diverse sets of regulatory elements and aid the interpretation of disease-associated genetic variation.
Dustin Shigaki, Orit Adato, Aashish N. Adhikari, Shengcheng Dong, Alex Hawkins-Hooker, Fumitaka Inoue, Tamar Juven-Gershon, Henry Kenlay, Beth Martin, Ayoti Patra, Dmitry D. Penzar, Max Schubach, Chenling Xiong, Zhongxia Yan, Alan P. Boyle, Anat Kreimer, Ivan V. Kulakovskiy, John Reid, Ron Unger, Nir Yosef, Jay Shendure, Nadav Ahituv, Martin Kircher, Michael A. Beer. Integration of multiple epigenomic marks improves prediction of variant impact in saturation mutagenesis reporter assay. 2019-06-23. https://doi.org/10.1002/humu.23797
A saturation-mutagenized 274-bp human HBG1 promoter library was assayed in HEL 92.1.7 erythroleukemia cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
The shared saturation-mutagenized 258-bp human TERT promoter library was assayed in the primary human glioblastoma cell culture SF7996 with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 578-bp human ZFAND3 enhancer library was assayed in MIN6 mouse pancreatic beta cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 384-bp human GP1BB promoter library was assayed in HEL 92.1.7 erythroleukemia cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 187-bp human HBB promoter library was assayed in HEL 92.1.7 erythroleukemia cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 285-bp human HNF4A promoter library was assayed in HEK293T cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 318-bp human LDLR promoter library was assayed in HepG2 cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 600-bp human IRF6 enhancer library was assayed in HaCaT keratinocytes with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 469-bp human PKLR promoter library was assayed in K562 cells with three independent plasmid transfections and a 48-hour harvest condition. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 591-bp human MSMB promoter library was assayed in HEK293T cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 600-bp human MYC enhancer sequence centered on the rs6983267 locus was assayed in HEK293T cells with three independent plasmid transfections. The condition included LiCl after the first 24 h and was harvested at 32 h.
A saturation-mutagenized 451-bp human IRF4 enhancer library was assayed in SK-MEL-28 melanoma cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 300-bp human F9 promoter library was assayed in HepG2 cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
The shared saturation-mutagenized 258-bp human TERT promoter library was assayed in HEK293T cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
A saturation-mutagenized 600-bp human SORT1 enhancer library was assayed in HepG2 cells with three independent plasmid transfections. Variant reporter activity was estimated from matched RNA and DNA tag counts.
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 20 files (ZIP)geo_variant_assignments/GSM3604135_F9.variants.txt.gzgeo_variant_assignments/GSM3604137_GP1BB.variants.txt.gzgeo_variant_assignments/GSM3604138_HBB.variants.txt.gzgeo_variant_assignments/GSM3604139_HBG1.variants.txt.gzgeo_variant_assignments/GSM3604140_HNF4A.variants.txt.gzgeo_variant_assignments/GSM3604141_IRF4.variants.txt.gzgeo_variant_assignments/GSM3604142_IRF6.variants.txt.gzgeo_variant_assignments/GSM3604144_LDLR.variants.txt.gzgeo_variant_assignments/GSM3604145_MSMB.variants.txt.gzgeo_variant_assignments/GSM3604147_MYCrs6983267.variants.txt.gzgeo_variant_assignments/GSM3604148_PKLR.variants.txt.gzgeo_variant_assignments/GSM3604152_SORT1.variants.txt.gzgeo_variant_assignments/GSM3604154_TERT.variants.txt.gzgeo_variant_assignments/GSM3604156_ZFAND3.variants.txt.gzGSE126550_filelist.txtREADME.txtsatMutMPRA_elements.tsv.gzsatMutMPRA_enhancers.tsvsatMutMPRA_promoters.tsvtarget_experiment_map.tsv