Figure 1.

Overview of LSV-seq for targeted detection of alternative splicing. (A) LSV-seq enriches for junction spanning reads by performing highly multiplexed RT with primers anchored directly adjacent to target LSVs. In contrast, only a minor fraction of conventional RNA-seq data is informative for splicing quantification, as most reads do not span splice junctions. (B) LSV formulation for splicing events used by the MAJIQ algorithm. Pie chart depicts the percentage of all splice junctions that can be captured by target LSVs, out of the superset of junctions in all source and target LSVs. LSV-seq primers can capture both classical binary splicing events as well as more complex events consisting of annotated and novel junctions. (C) Overview of LSV-seq protocol steps. LSV-seq targeting primers are first annealed to the target RNA using a touchdown protocol to maximize specificity. After first and second strand cDNA synthesis, linear amplification occurs via IVT. The amplified RNA (aRNA) then undergoes another RT step to append the second adapter. The resulting cDNA is then PCR-amplified and sequenced.