High Precision Detection of Rare Splice Isoforms Using Multiplexed Primer Extension Sequencing

Abstract

10Targeted RNA-sequencing aims to focus coverage on areas of interest that are inadequately 11 sampled in standard RNA-sequencing experiments. Here we present a novel approach for 12 targeted RNA-sequencing that uses complex pools of reverse transcription primers to enable 13 sequencing enrichment at user-selected locations across the genome. We demonstrate this 14 approach by targeting pre-mRNA splice junctions in S. cerevisiae, revealing high-precision 15 detection of splice isoforms, including rare pre-mRNA splicing intermediates. 16 17 3 Main text 18RNA sequencing (RNA-Seq) has greatly expanded our understanding of the variety of 19 splice isoforms that can be generated within a cell. Identification of the small subset of reads that 20 span exon-exon junctions within transcripts has enabled the unambiguous detection of vast 21 numbers of novel splice isoforms in scores of organisms 1,2 . Yet in spite of the power presented 22 elevated temperatures during the reverse transcription reaction minimizes non-specific primer 41 annealing (FigS1), while the inclusion of derivatized nucleotides allows for the efficient 42 purification of extended products and removal of excess primers. Secondly, a strand-extension 43 step similar to template-switching 7 is used to append the second sequencing adapter onto the 3' 44 terminus of the cDNA molecules. Coupling this approach with paired-end sequencing allows for 45 the simultaneous querying of the 5' and 3' ends of the cDNAs from targeted regions (see 46Methods for full details). 47