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RNA Sequencing Analysis Using Tophat

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RNA Sequencing Analysis Using Tophat Technical Note: RNA Analysis RNA Sequencing Analysis Using TopHat The Tuxedo Suite, comprising Bowtie, TopHat, and Cuffl inks, is widely adopted for RNA sequencing analysis, and can be run in multiple modes. Tuxedo Suite Galaxy The Tuxedo suite is a free, open-source collection of applications Galaxy (galaxy.psu.edu) is a web-based, open-source workfl ow that has been widely adopted as analysis toolset for differential gene manager with a thriving user base, documentation videos, and public expression, discovery of splice forms, and fast alignment of short forums for community questions4,5. Galaxy provides a workfl ow for reads. Some of the applications used in RNA sequencing analysis are running TopHat and associated applications from the Tuxedo suite: the following: https://main.g2.bx.psu.edu/root?tool_id=tophat • Bowtie1 is an ultrafast, memory-effi cient aligner for large sets of short reads. A good tutorial running TopHat through Galaxy is available here: TopHat2 • uses using Bowtie to align RNA-Seq reads to mammalian- https://main.g2.bx.psu.edu/u/jeremy/p/galaxy-rna-seq-analysis- sized genomes, and then analyzes the mapping results to identify exercise splice junctions between exons. Galaxy can be run using the free public server, but if you plan on • Cuffl inks3 assembles transcripts, estimates their abundances, and analyzing large datasets or sensitive samples, it may be better to tests for differential expression and regulation in RNA-Seq samples. install your own Galaxy instance, either locally or on the cloud. This is The RNA Sequencing analysis can either be run using the command explained on the following pages: line or the workfl ow manager Galaxy. http://wiki.g2.bx.psu.edu/Admin/Get Galaxy Command Line References The various applications in the Tuxedo suite all have their instructions 1. Langmead B, Trapnell C, Pop M, Salzberg SL. (2009) Ultrafast and memory- for using the command-line interface. You can use these, but you will effi cient alignment of short DNA sequences to the human genome. Genome need to understand where one application ends and the other starts. Biol 10(3):R25. To make it easy for you, we have generated a guide that covers the 2. Trapnell C, Pachter L, Salzberg SL.(2009) TopHat: discovering splice junc- entire workfl ow for RNA Sequencing, which can be downloaded from tions with RNA-Seq. Trapnell C, Pachter L, Salzberg SL. Bioinformatics Illumina’s website: 25(9):1105-11. http://www.illumina.com/documents/products/technotes/RNASeqA- 3. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, nalysisTopHat.pdf Salzberg SL, Wold BJ, Pachter L. (2010) Transcript assembly and quantifi - cation by RNA-Seq reveals unannotated transcripts and isoform switching Before running TopHat from the command line, you need to install the during cell differentiation. Nat Biotechnol 28(5):511-5. various applications on one of your servers. The same TopHat User 4. Langmead B, Trapnell C, and S Salzberg. (2009) Ultrafast and memoryef- Guide also provides installation instructions for all the applications in fi cient alignment of short DNA sequences to the human genome. Genome one place. Biol 10:R25. 5. Goecks, J, Nekrutenko, A, Taylor, J and The Galaxy Team. 2010 Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 11(8):R86. Illumina, Inc. • 9885 Towne Centre Drive, San Diego, CA 92121 USA • 1.800.809.4566 toll-free • 1.858.202.4566 tel • [email protected] • illumina.com FOR RESEARCH USE ONLY © 2012 Illumina, Inc. All rights reserved. Illumina, illuminaDx, Solexa, Making Sense Out of Life, Oligator, Sentrix, GoldenGate, GoldenGate Indexing, DASL, BeadArray, Array of Arrays, Infi nium, BeadXpress, VeraCode, IntelliHyb, iSelect, CSPro, GenomeStudio, Genetic Energy, and HiSeq are registered trademarks or trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Pub. No. 470-2012-001. Current as of 07 June 2012.
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