title: PrimerSeq: Design and visualization of RT-PCR primers for alternative splicing using RNA-seq data.
abstract: 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 large-scale 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.
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Collin
authors.initials: C
authors.lastname: Tokheim
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Juw Won
authors.initials: JW
authors.lastname: Park
authors.affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: yxing@ucla.edu.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xing
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017423:Sequence Analysis, RNA / Q000379:methods; D012984:Software
chemical_list: C034477:RNA primers; D012313:RNA
keywords: Alternative splicing; Primer design; RNA-seq; Transcriptome; Visualization
references.citation: BMC Bioinformatics. 2012 Apr 19;13 Suppl 6:S5
references.pmid: 22537044
references.citation: RNA. 2010 Feb;16(2):442-9
references.pmid: 20038630
references.citation: Nucleic Acids Res. 2011 May;39(9):3820-35
references.pmid: 21245033
references.citation: Database (Oxford). 2011 Sep 14;2011:bar040
references.pmid: 21917859
references.citation: Nature. 2010 Jan 28;463(7280):457-63
references.pmid: 20110989
references.citation: Nat Biotechnol. 2010 May;28(5):503-10
references.pmid: 20436462
references.citation: Nat Rev Genet. 2007 Oct;8(10):749-61
references.pmid: 17726481
references.citation: Proc Natl Acad Sci U S A. 2011 Feb 15;108(7):2837-42
references.pmid: 21282640
references.citation: Bioinformatics. 2004 Oct 12;20(15):2471-2
references.pmid: 15073005
references.citation: Nat Struct Mol Biol. 2009 Jun;16(6):670-6
references.pmid: 19448617
references.citation: BMC Bioinformatics. 2008 Nov 01;9:465
references.pmid: 18976492
references.citation: Methods. 2014 Apr 1;66(3):398-409
references.pmid: 23792920
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D942-5
references.pmid: 18948285
references.citation: BMC Bioinformatics. 2008 May 29;9:253
references.pmid: 18510760
references.citation: Proc Natl Acad Sci U S A. 2013 Jul 30;110(31):12750-5
references.pmid: 23852726
references.citation: Genome Res. 2004 Mar;14(3):426-41
references.pmid: 14962984
references.citation: Bioinformatics. 2002;18 Suppl 1:S181-8
references.pmid: 12169546
references.citation: Brief Bioinform. 2013 Mar;14(2):144-61
references.pmid: 22908213
references.citation: Nat Biotechnol. 2010 May;28(5):511-5
references.pmid: 20436464
references.citation: Mol Cell Biol. 2012 Apr;32(8):1468-82
references.pmid: 22354987
references.citation: Nat Methods. 2010 Dec;7(12):1009-15
references.pmid: 21057496
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D805-9
references.pmid: 17068075
references.citation: Nucleic Acids Res. 2012 Apr;40(8):e61
references.pmid: 22266656
references.citation: Nucleic Acids Res. 2012 Aug;40(15):e115
references.pmid: 22730293
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D1144-9
references.pmid: 22086960
references.citation: PLoS Genet. 2008 Oct 17;4(10):e1000225
references.pmid: 18841251
references.citation: RNA. 2012 Jul;18(7):1395-407
references.pmid: 22627775
references.citation: Nat Rev Genet. 2011 Sep 16;12(10):715-29
references.pmid: 21921927
references.citation: Nucleic Acids Res. 2006 Jun 06;34(10):3150-60
references.pmid: 16757580