Detection of splice isoforms and rare intermediates using multiplexed primer extension sequencing

Targeted RNA-sequencing aims to focus coverage on areas of interest that are inadequately sampled in standard RNA-sequencing experiments. Here we present a novel approach for targeted RNA-sequencing that uses complex pools of reverse transcription primers to enable sequencing enrichment at user-selected locations across the genome. We demonstrate this approach by targeting pre-mRNA splice junctions in S. cerevisiae, revealing high-precision detection of splice isoforms, including rare pre-mRNA splicing intermediates.