PrimerSeq: Design and Visualization of RT-PCR Primers for Alternative Splicing Using RNA-seq Data

The vast majority of multi-exon genes in higher eukaryotes are alternatively spliced and changes in alternative splicing（AS） can impact gene function or cause disease. High-throughput RNA sequencing（RNA-seq） has become a powerful technology for transcriptome-wide analysis of AS, but RT-PCR still remains the gold-standard approach for quantifying and validating exon splicing levels. We have developed PrimerSeq, a user-friendly software for systematic design and visualization of RT-PCR primers using RNA-seq data. PrimerSeq incorporates user-provided transcriptome profiles（i.e., RNA-seq data） in the design process, and is particularly useful for largescale quantitative analysis of AS events discovered from RNA-seq experiments. PrimerSeq features a graphical user interface（GUI） that displays the RNA-seq data juxtaposed with the expected RT-PCR results. To enable primer design and visualization on user-provided RNA-seq data and transcript annotations, we have developed PrimerSeq as a stand-alone software that runs on local computers. PrimerSeq is freely available for Windows and Mac OS X along with source code at http：//primerseq.sourceforge.net/. With the growing popularity of RNA-seq for transcriptome studies, we expect PrimerSeq to help bridge the gap between high-throughput RNA-seq discovery of AS events and molecular analysis of candidate events by RT-PCR.