Post on 12-Feb-2022
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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.
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Outline
• 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
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Hot Topics: RNA-seq Analysis in Galaxy.
The Galaxy Interface A web based platform for analysis of large genomic datasets
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within one interface.
an analysis. Even if you delete the datasets, the history keeps the tools used.
No need of programming experience.
Integrates many bioinformatics tools within one interface.
Keeps track of all the steps performed in an analysis. Even if you delete the datasets, the history keeps the tools used.
LOCAL COPY
storage
Jobs are sent to the cluster
LOCAL COPY
Faster Customizable 250Gb of storage Data is private Jobs are sent to the cluster
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)
Hot Topics: RNA-seq Analysis in Galaxy.
Galaxy Interface: Analyze Data
Tools window
Data display and tool’s dialog window
History window:
Data analysis
Processed data Green: job is finished Yellow: job is running Gray: job is in queue Red: there is a problem
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History window:
History window: All analysis steps are saved. Data is not overwritten. Can create workflow to repeat an analysis.
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Hot Topics: RNA-seq Analysis in Galaxy.
How to find your previous histories
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History menu
RNA-seq Experiment
Wang, Z. et al. RNA-Seq: a revolutionary tool for transcriptomics Nature Reviews Genetics (2009)
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Hot Topics: RNA-seq Analysis in Galaxy.
RNA-seq Applications
• Annotation
Identify novel genes, transcripts, exons, splicing events, ncRNAs.
• Detecting RNA editing and SNPs.
• Measurements: RNA quantification and differential gene expression
Abundance of transcripts between different conditions
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Hot Topics: RNA-seq Analysis in Galaxy. Hot Topics: RNA-seq Analysis in Galaxy.
Getting Data: Uploading Large Files Step 1: copy your file to
/nfs/galaxy/uploads/username@wi.mit.edu using a sftp client
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CyberDuck
/nfs/galaxy/uploads/username@wi.mit.edu
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Hot Topics: RNA-seq Analysis in Galaxy.
Getting Data: Uploading Large Files Step 2: Select and upload the file within galaxy
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Execute
Genome Assembly
Upload Fie
Preprocessing NGS Quality Control: FastQC
Hot Topics: RNA-seq Analysis in Galaxy.
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Hot Topics: RNA-seq Analysis in Galaxy.
Preprocessing: Remove bad quality reads FASTX-TOOLKIT ->Filter by quality
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RNA-seq Analysis: Tuxedo Tools
Hot Topics: RNA-seq Analysis in Galaxy.
Tool Tool description
Bowtie Ultrafast short read aligner
Tophat Aligns RNA-seq reads to the genome using Bowtie. Discovers splice sites
Cufflinks package
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
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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
Hot Topics: RNA-seq Analysis in Galaxy.
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Tutorials and References • Galaxy tutorials
http://galaxy.psu.edu/screencasts.html
• Previous Hot Topics
http://jura.wi.mit.edu/bio/education/hot_topics
• SOPs (Standard operating procedures)
https://gir.wi.mit.edu/trac/wiki/barc/SOPs
• 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
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