This 2-day workshop, which includes both lecture and hands-on components, will cover the fundamentals of and best practices for RNA-Seq Data Analysis. Learn everything from experimental design and sample prep requirements, to alignment, quantification, generation of differentially expressed genes, and understanding the results after completion of analysis. There will be presentations and training on using both open source (CCBR RNA-Seq Pipeline) and commercial (Partek Flow) software.
NOTE: This is a BYOC (Bring your own laptop Computer) class. Government issued or personal computers are permitted. We will be able to supply a very limited set of computers, so if you want to take the class but cannot bring your own computer please indicate such in the Comment section on the registration form.
WORKSHOP AGENDA
Monday, March 20 - Morning Session
9:35 - 10:35 am Introduction to RNA-Seq
Speaker: Maggie Cam, Ph.D.
The first part of the session will provide an introduction to the technology, its various applications, comparison to microarray, advantages as well as limitations, and costs associated with doing this NGS experiment. The second part of the talk will focus on best practices to be adopted when considering RNA-Seq for your research, including sample quality, sequencing depth, replicates, analytical methods, and results generated with this approach.
10:40 - 11:40 am Introduction to Single Cell RNA-Seq
Speaker: Michael Kelly, PhD
This presentation will discuss how scRNASeq is different from conventional RNA-Seq, special challenges, specific applications, experimental and analytical requirements.
11:45 am -12:30 pm Understanding the NGS vocabulary and file formats
Speaker: Peter C. Fitzgerald, PhD
This talk will discuss file formats and terms commonly used for NGS data analysis.
LUNCH BREAK 12:30 - 1:00 pm
Monday, March 20 - Afternoon Session
1:00 - 4:00 pm CCBR RNA-Seq Pipeline
Speaker: Fathi Elloumi, PhD
This session will be a comprehensive review of the pipeline built and used by CCBR for analysis of RNA-Seq data, and will cover:
1. Introduction
2. Analytical Steps
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Initial QC
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Alignment - aligners, transcriptome vs genome, non-aligners
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Post-Alignment QC
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Expression quantitation, FPKM, counts, Junctions reads
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Normalization (different methods)
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Differential Expression gene/transcriptome lists
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Isoform calling
3. Demo on the RNA-Seq pipeline
4. Detailed review of pre-analyzed results
- Multi-QC results
- Differentially Expressed Genes
- Isoform Expression (high-level review)
5. Next Steps
- Viewers - Manual validation (IGV)
- Principal Component Analysis (PCA)
- Clustering - Different methods
- Pathway Enrichment (GSEA)
6. Final, brief hands-on session for ONLY those who fulfill the following requirements:
- Already have an existing Biowulf/Helix Account
- Basic working knowledge of command line on Terminal
- Functional XQuartz application (https://www.xquartz.org/releases/XQuartz-2.7.11.html)
This is applicable for only this last session on Day 1. Not necessary for other sessions on Day 1 or Day 2.
Day 2 - Tuesday, March 21
9:30 am - 10:00 am Set up time for Partek Flow and Partek Genomics Suite
10:00 - 12:30 pm RNA-Seq Analysis using Partek Flow
Presenter: Eric Seiser, PhD - Partek Field Application Specialist
An overview of getting started on the NIH Helix server and then a live demo of RNA-seq analysis in Partek Flow. The training will highlight key concepts in RNA-seq analysis and their implementation Flow. This will be followed by a demo utilizing Partek Flow to importing raw sequence data in fastq format from a published study, followed by performing QA/QC, alignment, quantification, differential expression detection and finally biological interpretation.
LUNCH BREAK 12:30 - 1:00 pm
1:00 - 4:00 pm Read count data analysis using Partek Flow and Genomics Suite
Presenter: Eric Seiser, PhD - Partek Field Application Specialist
This second part of the training session will allow users to take raw RNA-seq data from a recently published study and independently build a complete analysis pipeline within Partek Flow, allowing students to ask questions as they analyze the data. This will be followed by an overview of RNA-seq functionality in Partek Genomics Suite, focusing on plotting options, data integration, and enrichment analysis. The final part of the training will allow users hand-on training to import the results from their Flow analysis pipeline and explore the downstream features in Genomics Suite. This time will also be used as a general question and answer session.
Students will learn how to use basic features of Partek Flow and Partek Genomics Suite, including:
· Partek Flow
• Getting set up on NIH Helix server
• Importing data
• Performing QA/AC
• Alignment
• Gene/transcript abundance estimation
• Differential expression detection
• Go Enrichment analysis
• Visualization (PCA, dotplot, volcano plot, chromosome view, hierarchical clustering etc.)
• Microarray analysis and integration with RNA-seq data.
· Partek Genomics Suite
• Importing Partek Flow project and text file format
• Visualization (PCA, dot plot, heatmap etc.)
• Pathway analysis
• Integration of genomic data.
* Instructions for setting up access to Partek Flow will be provided to attendees at the workshop.
**Information for installing Partek Genomics Suite will be sent to registrants ahead of the workshop.
