GSE126583
Multiplexed primer extension sequencing: A targeted RNA-seq method that enables high-precision quantitation of mRNA splicing isoforms and rare pre-mRNA splicing intermediates
The study of splicing has been greatly aided by the advent of RNA sequencing (RNA-seq), which can enable the genome-wide detection of discrete splice isoforms. Quantification of these splice isoforms requires analysis of splicing informative sequencing reads, those that unambiguously map to a single splice isoform, including exon-intron spanning reads corresponding to retained introns, as well as exon-exon junction spanning reads corresponding to either canonically- or alternatively-spliced isoforms. Because most RNA-seq experiments are designed to produce sequencing reads that uniformly cover the entirety of transcripts, only a comparatively small number of splicing informative reads are generated for any given splice site, leading to a decreased ability to detect and/or robustly quantify less abundant isoforms. To address this problem, we have recently described a method termed Multiplexed Primer Extension sequencing, or MPE-seq, which uses complex pools of thousands of reverse transcription primers to target sequencing to user selected loci. By targeting reverse transcription to pre-mRNA splice junctions, this approach can enable a dramatic enrichment in the fraction of splicing informative reads generated in an experiment, enabling an increase both in the precision with which splicing efficiency can be measured, and in the detection of splice isoforms including rare splicing intermediates. Here we provide a brief review of methods for calculating splicing efficiency with existing RNA-seq data, including the shortcomings associated with these approaches that drove our development of MPE-seq, as well as a detailed protocol for implementation of MPE-seq. Overall design: MPE-seq of two WT S. pombe samples