Understanding the functional impact of genomic variants is a major goal of modern genetics and personalized medicine. Although many synonymous and non-coding variants act through altering the efficiency of pre-mRNA splicing, it is difficult to predict how these variants impact pre-mRNA splicing.
Researchers from UConn Health describe a massively parallel approach they use to test the impact on pre-mRNA splicing of 2059 human genetic variants spanning 110 alternative exons. This method, called variant exon sequencing (Vex-seq), yields data that reinforce known mechanisms of pre-mRNA splicing, identifies variants that impact pre-mRNA splicing, and will be useful for increasing our understanding of genome function.
Assembly of test exon and experimental design
a The test exon and flanking introns are subcloned into a reporter plasmid in a two-step process, such that the barcode designating the sequence is near the end of the transcript. Once these plasmids are transfected into cultured cells, a transcript will be produced that may not contain the variant itself, but does contain the barcode (b) uniquely associated with the variant tested. A ten-nucleotide UMI (N10) is attached during the reverse transcription step to collapse PCR duplicates downstream. Illumina flow cell binding sequences (FC) and indexes (I1 and I2) are attached via primers during PCR and the resulting DNA is sequenced on a MiSeq platform. b Data analysis pipeline for splicing results