title: Detection and visualization of differential splicing in RNA-Seq data with JunctionSeq.
abstract: Although RNA-Seq data provide unprecedented isoform-level expression information, detection of alternative isoform regulation (AIR) remains difficult, particularly when working with an incomplete transcript annotation. We introduce JunctionSeq, a new method that builds on the statistical techniques used by the well-established DEXSeq package to detect differential usage of both exonic regions and splice junctions. In particular, JunctionSeq is capable of detecting differential usage of novel splice junctions without the need for an additional isoform assembly step, greatly improving performance when the available transcript annotation is flawed or incomplete. JunctionSeq also provides a powerful and streamlined visualization toolset that allows bioinformaticians to quickly and intuitively interpret their results. We tested our method on publicly available data from several experiments performed on the rat pineal gland and Toxoplasma gondii, successfully detecting known and previously validated AIR genes in 19 out of 19 gene-level hypothesis tests. Due to its ability to query novel splice sites, JunctionSeq is still able to detect these differences even when all alternative isoforms for these genes were not included in the transcript annotation. JunctionSeq thus provides a powerful method for detecting alternative isoform regulation even with low-quality annotations. An implementation of JunctionSeq is available as an R/Bioconductor package.
authors.affiliation: Comparative Genomics Analysis Unit, Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA stephen.hartley@nih.gov.
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Hartley
authors.affiliation: Comparative Genomics Analysis Unit, Cancer Genetics and Comparative Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: James C
authors.initials: JC
authors.lastname: Mullikin
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D002940:Circadian Rhythm / Q000235:genetics; D003196:Computer Graphics; D066264:Datasets as Topic; D005091:Exons / Q000235:genetics; D006801:Humans; D058977:Molecular Sequence Annotation; D010870:Pineal Gland / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics*; D051381:Rats; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA / Q000379:methods*; D014122:Toxoplasma / Q000235:genetics
chemical_list: D020033:Protein Isoforms; D022821:RNA Splice Sites