We developed PASSPORT-seq to screen 100s of SNPs that are predicted to alter miRNA–mRNA interactions. The availability of pooled oligonucleotide synthesis and the NEBuilder® gene assembly system have made this assay possible. Our assay builds and substantially improves the technologies that have had only limited success (Reid et al., 2009). For example, in previous high-throughput splicing assays, short inserts were underrepresented during the library construction using traditional cloning methods (Chen and Chasin, 1994; Ke et al., 2011; Soemedi et al., 2014). In contrast, our library had representation of all allele pairs. This may be explained by the cloning method we utilized, i.e., the NEBuilder® gene assembly system that produces covalently closed circular plasmids as opposed to traditional cloning methods, which yield a nicked-relaxed plasmid topology. This change in topology may result in a more efficient plasmid uptake by chemically competent bacteria (Hanahan, 1983; Xie et al., 1992; Kobori and Nojima, 1993). Better transformation efficiency increases the probability of both the variant and reference plasmids being represented in the resulting pool, which is a critical prerequisite for studying the allele-specific activity of miRNAs.
