Download RNA-seq Analysis in Galaxy

1/17/2013
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Outline
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RNA-seq Analysis in Galaxy
Local copy of Galaxy: https://galaxy.wi.mit.edu/
Main site: http://main.g2.bx.psu.edu/
January, 2013
Hot Topics: RNA-seq Analysis in Galaxy.
What is the Galaxy Interface?
What is RNA-seq?
Data upload in Galaxy
Data preprocessing in Galaxy
Tuxedo tools for RNA-seq analysis
RNA-seq analysis workflows: Hands-on
Hot Topics: RNA-seq Analysis in Galaxy.
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The Galaxy Interface
A web based platform for analysis of large genomic datasets
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Galaxy Interface: Analyze Data
Data analysis
Type “https://galaxy.wi.mit.edu/”
in your browser address.
You will be prompted for your
name and password (these are the
same that you use for your email)
Processed data
Green:
job is finished
Yellow:
job is running
Gray:
job is in queue
LOCAL COPY
 need of programming experience.
No
Integrates many bioinformatics tools
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within one interface.
Keeps track of all the steps performed in
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an analysis. Even if you delete the datasets,
the history keeps the tools used.
Faster
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Customizable
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250Gb
of
storage
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Data
is private
Jobs are sent to
the cluster
Red: there is a
problem
Tools
window
History window:
All analysis steps are saved.
Data is not
overwritten.
History window:
Data display and tool’s dialog
Can create workflow to repeat an
window
analysis.
Hot Topics: RNA-seq Analysis in Galaxy.
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RNA-seq Experiment
How to find your previous histories
History
menu
Wang, Z. et al. RNA-Seq: a revolutionary tool for transcriptomics Nature Reviews Genetics (2009)
Hot Topics: RNA-seq Analysis in Galaxy.
Hot Topics: RNA-seq Analysis in Galaxy.
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RNA-seq Applications
• Annotation
Getting Data: Uploading Large Files
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Step 1: copy your file to
/nfs/galaxy/uploads/[email protected]
using a sftp client
Identify novel genes, transcripts, exons, splicing
events, ncRNAs.
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• Detecting RNA editing and SNPs.
• Measurements: RNA quantification and
differential gene expression
CyberDuck
Abundance of transcripts between different
conditions
/nfs/galaxy/uploads/[email protected]
Hot Topics: RNA-seq Analysis in Galaxy.
Hot Topics: RNA-seq Analysis in Galaxy.
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Getting Data: Uploading Large Files
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Preprocessing
NGS Quality Control: FastQC
Step 2: Select and upload the file within galaxy
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Upload Fie
Execute
Genome
Assembly
Hot Topics: RNA-seq Analysis in Galaxy.
Hot Topics: RNA-seq Analysis in Galaxy.
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Preprocessing: Remove bad quality reads
FASTX-TOOLKIT ->Filter by quality
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RNA-seq Analysis: Tuxedo Tools
Tool
Cufflinks
package
Hot Topics: RNA-seq Analysis in Galaxy.
Tool description
Bowtie
Ultrafast short read aligner
Tophat
Aligns RNA-seq reads to the genome using
Bowtie. Discovers splice sites
Cufflinks
Assembles transcripts
Cuffcompare
Compares transcript assemblies to annotation
Cuffmerge
Merges two or more transcript assemblies
Cuffdiff
Finds differentially expressed genes and
transcripts. Detects differential splicing and
promoter use
Hot Topics: RNA-seq Analysis in Galaxy.
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Examples of analysis workflows
Hands-on 1
Differential expression
analysis
Hands-on 2
Transcript assembly and
differential expression analysis
Hands-on 3
Transcript assembly and
transcript comparison
Tutorials and References
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Galaxy tutorials
http://galaxy.psu.edu/screencasts.html
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Previous Hot Topics
http://jura.wi.mit.edu/bio/education/hot_topics
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SOPs (Standard operating procedures)
https://gir.wi.mit.edu/trac/wiki/barc/SOPs
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References
Taylor et al. (2007) Using Galaxy to perform large-scale interactive data analyses. Current
Protocols in Bioinformatics Chapter 10, unit 10.
Blankenberg et al. (2010) Manipulation of FASTQ data with Galaxy. Bioinformatics
26(14):1783-5
Trapnell et al. (2012) Differential gene and transcript expression analysis of RNA-seq
experiments with TopHat and Cufflinks. Nature Protocols 7, 562–578
Tophat Manual: http://tophat.cbcb.umd.edu/manual.html
Cufflinks Manual: http://cufflinks.cbcb.umd.edu/manual.html
Hot Topics: RNA-seq Analysis in Galaxy.
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