RNA isoform-resolved multiplexed sequencing with bioorthogonal barcoding

RNA isoform dysregulation is a significant cause of disease pathogenesis and has been targeted by FDA-approved splice-switching therapeutics. However, current multiplexed sequencing methods fail to preserve splice junction information as they only barcode and count three termini. To address this limitation, researchers have devised a method to attach bioorthogonal barcodes (bobcodes) to multiple internal positions on cellular RNAs using acylation and click chemistries.

These bobcoded RNAs can be pooled for multiplexed cDNA synthesis, during which reverse transcriptase accurately switches between each RNA template and its tethered bobcode with greater than 99% accuracy in species mixing experiments. By strategically attaching bobcodes at specific intervals, researchers set the length of cDNA inserts without the need for a library fragmentation step.

Priming with either poly(dT) or random hexamers allows for either 3'-end counting or full-length isoform capture. This innovative approach, dubbed bioorthogonal barcode-sequencing (BOB-seq v0.1), enables a drug screen that identifies transcriptome-wide on- and off-target RNA splicing effects while outperforming existing multiplexing RNA sequencing methods in workflow simplicity, sample-to-sample variability, and barcoding accuracy.

The incorporation of bobcodes adds isoform resolution to scalable multiplexed RNA sequencing, offering a valuable tool for studying RNA splicing and its role in disease.