Cynthia A. Kalita, Christopher D. Brown, Andrew Freiman, Jenna Isherwood, Xiaoquan Wen et al.
Many variants associated with complex traits are in noncoding regions and contribute to phenotypes by disrupting regulatory sequences. To characterize these variants, we developed a streamlined protocol for a high-throughput reporter assay, Biallelic Targeted STARR-seq (BiT-STARR-seq), that identifies allele-specific expression (ASE) while accounting for PCR duplicates through unique molecular identifiers. We tested 75,501 oligos (43,500 SNPs) and identified 2720 SNPs with significant ASE (FDR < 10%). To validate disruption of binding as one of the mechanisms underlying ASE, we developed a new high-throughput allele-specific binding assay for NFKB1. We identified 2684 SNPs with allele-specific binding (ASB) (FDR < 10%); 256 of these SNPs also had ASE (OR = 1.97, P-value = 0.0006). Of variants associated with complex traits, 1531 resulted in ASE, and 1662 showed ASB. For example, we characterized that the Crohn's disease risk variant for rs3810936 increases NFKB1 binding and results in altered gene expression.
Cynthia A. Kalita, Christopher D. Brown, Andrew Freiman, Jenna Isherwood, Xiaoquan Wen, Roger Pique-Regi, Francesca Luca. High-throughput characterization of genetic effects on DNA–protein binding and gene transcription. 2018-09-25. https://doi.org/10.1101/gr.237354.118
A biallelic targeted STARR-seq library containing 75,501 published direction-specific result records representing 43,500 SNPs was tested in the GM18507 human lymphoblastoid cell line. Nine biological transfections and seven DNA-library replicates were analyzed with UMI-aware QuASAR-MPRA to measure allele-specific reporter expression relative to the input plasmid allele proportion.
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Download all 15 files (ZIP)source_notes.txtsra_experiment_packages.xmlsupplemental_methods_bit_starr_bundle.pdfsupplemental_methods_plasmid_dna.zipsupplemental_table_S10_bundle_deseq_high.txtsupplemental_table_S11_bundle_deltaast_results.txtsupplemental_table_S14_bundle_complex_traits.txtsupplemental_table_S15_ase_complex_traits.txtsupplemental_table_S1_bit_starr_results.txtsupplemental_table_S2_centisnp_annotations.txtsupplemental_table_S3_gwas_annotations.txtsupplemental_table_S4_eqtl_annotations.txtsupplemental_table_S7_bundle_deseq_combined.txtsupplemental_table_S8_bundle_deseq_low.txtsupplemental_table_S9_bundle_deseq_mid.txt