Tunc Morova, Yi Ding, Chia-Chi F. Huang, Funda Sar, Tommer Schwarz et al.
The vast majority of disease-associated single nucleotide polymorphisms (SNP) identified from genome-wide association studies (GWAS) are localized in non-coding regions. A significant fraction of these variants impact transcription factors binding to enhancer elements and alter gene expression. To functionally interrogate the activity of such variants we developed snpSTARRseq, a high-throughput experimental method that can interrogate the functional impact of hundreds to thousands of non-coding variants on enhancer activity. snpSTARRseq dramatically improves signal-to-noise by utilizing a novel sequencing and bioinformatic approach that increases both insert size and the number of variants tested per loci. Using this strategy, we interrogated known prostate cancer (PCa) risk-associated loci and demonstrated that 35% of them harbor SNPs that significantly altered enhancer activity. Combining these results with chromosomal looping data we could identify interacting genes and provide a mechanism of action for 20 PCa GWAS risk regions. When benchmarked to orthogonal methods, snpSTARRseq showed a strong correlation with in vivo experimental allelic-imbalance studies whereas there was no correlation with predictive in silico approaches. Overall, snpSTARRseq provides an integrated experimental and computational framework to functionally test non-coding genetic variants.
Tunc Morova, Yi Ding, Chia-Chi F. Huang, Funda Sar, Tommer Schwarz, Claudia Giambartolomei, Sylvan C. Baca, Dennis Grishin, Faraz Hach, Alexander Gusev, Matthew L. Freedman, Bogdan Pasaniuc, Nathan A. Lack. Optimized high-throughput screening of non-coding variants identified from genome-wide association studies. 2022-12-22. https://doi.org/10.1093/nar/gkac1198
A capture-enriched human genomic-fragment library containing prostate-cancer-associated SNP loci and positive/negative control regions was transiently transfected into LNCaP prostate cancer cells in three biological replicas. Reporter RNA self-transcription was normalized to plasmid DNA input and reference-versus-alternative allele activity was tested with negative-binomial regression at the 24 h harvest.
A capture-enriched human genomic-fragment library containing prostate-cancer-associated SNP loci and positive/negative control regions was transiently transfected into LNCaP prostate cancer cells in three biological replicas. Reporter RNA self-transcription was normalized to plasmid DNA input and reference-versus-alternative allele activity was tested with negative-binomial regression at the 48 h harvest.
A capture-enriched human genomic-fragment library containing prostate-cancer-associated SNP loci and positive/negative control regions was transiently transfected into LNCaP prostate cancer cells in three biological replicas. Reporter RNA self-transcription was normalized to plasmid DNA input and reference-versus-alternative allele activity was tested with negative-binomial regression at the 72 h harvest.
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 12 files (ZIP)308.bedeuropepmc_supplementary_bundle.zipgkac1198_supplemental_files.zipGRCh37-v1.6_6123ca426476f4eb96fdfaa8519c34fd.tsvraw_results_merged_24h.txtraw_results_merged_48h.txtraw_results_merged_72h.txtREADME.txtsha256sums.txtsnpSTAR_supplementary_figures.pdfsnpSTARRseq_Supplementary_Tables.xlsxtotal500.bed