PASSPORT-seq: A Novel High-Throughput Bioassay to Functionally Test Polymorphisms in Micro-RNA Target Sites

Abstract

Next-generation sequencing (NGS) studies have identified large numbers of genetic variants that are predicted to alter miRNA–mRNA interactions. We developed a novel high-throughput bioassay, PASSPORT-seq, that can functionally test in parallel 100s of these variants in miRNA binding sites (mirSNPs). The results are highly reproducible across both technical and biological replicates. The utility of the bioassay was demonstrated by testing 100 mirSNPs in HEK293, HepG2, and HeLa cells. The results of several of the variants were validated in all three cell lines using traditional individual luciferase assays. Fifty-five mirSNPs were functional in at least one of three cell lines (FDR ≤ 0.05); 11, 36, and 27 of them were functional in HEK293, HepG2, and HeLa cells, respectively. Only four of the variants were functional in all three cell lines, which demonstrates the cell-type specific effects of mirSNPs and the importance of testing the mirSNPs in multiple cell lines. Using PASSPORT-seq, we functionally tested 111 variants in the 3′ UTR of 17 pharmacogenes that are predicted to alter miRNA regulation. Thirty-three of the variants tested were functional in at least one cell line.

Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders

“…Among the genes with AUDassociated differences in ASE in at least one of the four brain regions, we identified 565 SNPs in the 3′UTR regions; 437 SNPs in 61 genes had at least one heterozygote in either the AUD or control group (Supplementary Table 3). We adapted a high-throughput reporter assay, PASSPORT-seq [13], to identify 3′UTR variants that affected gene expression (RNA levels) in two human neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2) (Supplementary Table 4). We detected expression of the reference and alternative alleles in both SH-SY5Y and SK-N-BE(2) cells in 362 (82.8%) of the 437 SNPs screened.…”

Allele-Specific Expression and High-Throughput Reporter Assay Reveal Functional Variants in Human Brains with Alcohol Use Disorders

“…In this study, we used a high-throughput reporter assay, PASSPORT-seq [9], to screen for 3'UTR variants that lead to gene expression changes. We modified the original protocol to improve the quality of the data.…”

“…Among these, 437 SNPs in 61 genes had at least one heterozygote in either the AUD or control group (Supplementary Table 3). We adapted a high-throughput reporter assay, PASSPORT-seq [9], to identify 3'UTR variants that affect gene expression (RNA levels) in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE (2). A schematic of the overall procedure is shown in Figure 2.…”

“…The PASSPORT-seq assay was conducted as previously described [9] with some modifications. Briefly, the procedure includes the following steps: oligonucleotide synthesis, plasmid library construction, transfection, DNA/RNA extraction, and sequencing.…”

“…We integrated deep RNA-seq and single nucleotide polymorphism (SNP) genotyping data from four different brain regions of 30 subjects with AUD and 30 social/non-drinking control subjects to detect genes whose ASE differs. We then used a high-throughput reporter assay, PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) [9] to systematically screen 3' untranslated region (3'UTR) variants in those genes to determine which alter RNA levels in cells of neural origin. This assay offers a powerful tool to screen genomic variants associated with a specific phenotype to determine which affect gene regulation.…”