Alternative Splicing Signatures in RNA‐seq Data: Percent Spliced in (PSI)

Sebastian Schafer1, Kui Miao2, Craig C. Benson3, Matthias Heinig4, Stuart A. Cook5, Norbert Hubner6

1 National Heart Center Singapore, 2 Duke‐National University of Singapore, 3 Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, 4 Present address: Institute of Computational Biology, Helmholtz Zentrum München, Neuerberg, 5 National Heart and Lung Institute, Imperial College London, London, 6 Charité‐Universitätsmedizin, Berlin
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 11.16
DOI:  10.1002/0471142905.hg1116s87
Online Posting Date:  October, 2015
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Abstract

Thousands of alternative exons are spliced out of messenger RNA to increase protein diversity. High‐throughput sequencing of short cDNA fragments (RNA‐seq) generates a genome‐wide snapshot of these post‐transcriptional processes. RNA‐seq reads yield insights into the regulation of alternative splicing by revealing the usage of known or unknown splice sites as well as the expression level of exons. Constitutive exons are never covered by split alignments, whereas alternative exonic parts are located within highly expressed splicing junctions. The ratio between reads including or excluding exons, also known as percent spliced in index (PSI), indicates how efficiently sequences of interest are spliced into transcripts. This protocol describes a method to calculate the PSI without prior knowledge of splicing patterns. It provides a quantitative, global assessment of exon usage that can be integrated with other tools that identify differential isoform processing. Novel, complex splicing events along a genetic locus can be visualized in an exon‐centric manner and compared across conditions. © 2015 by John Wiley & Sons, Inc.

Keywords: alternative splicing; RNA‐seq; percent spliced in; PSI; transcript processing; isoform expression

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Calculation of Percent Spliced in for Annotated Exons
  • Alternate Protocol 1: Calculation of Percent Spliced in for Annotated Exons with the Script PSI.sh
  • Support Protocol 1: Create an Exonic Part Annotation Based on UCSC Gene Annotation Files
  • Support Protocol 2: Reformat STAR Aligner Output Data to Calculate the PSI
  • Commentary
  • Figures
     
 
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Materials

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Literature Cited

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Internet Resources
  https://github.com/arq5x/bedtools2
  BEDTools Suite Web site, which allows for downloading and installing the BEDTools applications. This toolset can analyze genome‐wide datasets and work with genomic intervals.
  http://genome.ucsc.edu/
  UCSC Genome browser Web site to download gene annotations and view genomic interval files.
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Supplementary Material