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Workshop Code of Conduct
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DIBSI Nonmodel mRNASeq Workshop (2017)
Workshop materials
Booting a Jetstream Computer Instance for your use!
Request to log in to the Jetstream Portal
Use “XSEDE”
Fill in the username and password and click “Sign in”
Select Projects and “Create New Project”
Name the project for yourself, click “create”
Select the newly created project
Within the project, select “new”
Find the “Ubuntu 16.04” image, click on it
Name it something simple and select ‘m1.medium’
Wait for it to become active
Click on your new instance to get more information!
Miscellany
Suspend your instance
Shutting down your instance
Deleting your instance
Logging in to jetstream from your local terminal with a key file
Concerning Keys
Getting your instance IP address
Setting up keys
Adding password to a Jetstream instance
Short read quality and trimming
Installing some software
Data source
Check that your data is where it should be
Quality trimming and light quality filtering
De novo transcriptome assembly with Trinity
Installation
Then, let’s check we still have our reads from yesterday’s QC lesson
Let’s do digital normalization with khmer
Applying Digital Normalization
Running the Actual Assembly!
Evaluating your transcriptome assembly
Install
Transrate
BUSCO
Annotating de novo transcriptomes with dammit
Installation
Database Preparation
Annotation
Parse dammit output
References
Analyzing RNAseq expression with Salmon
Getting Started
Installation
Download and make Salmon available in the path
Then, let’s check we still have our reads from yesterday’s QC work
Download or link an assembly
Run Salmon
Working with the counts
Other useful tutorials and references
Differential expression analysis with DESeq2
Upgrade R (3.4.x)
Make sure you’re running RStudio
Install RStudio Web server
Install DESeq2 prereqs
Move salmon output quant files to their own directory
Move the gene names to your home directory (to easily access it)
Grab a special script plotPCAWithSampleNames.R
RStudio!
Introduction to automation
Keeping a log of what you ran
While it’s running…
After it’s done running: put it in a shell script
Passing variables to a script
Long-running jobs more generally
Scripts: ‘bash’ shell scripts, R scripts, and Python scripts
Final thoughts
GitHub
The Dashboard
Making Repositories
Cloning Repositories
Commits
Issues
Forking and Pull Requests
GitHub Education
Using GitHub repositories to store your scripts
Create a new repository
Edit README.md
Clone your repository
Fork an existing repository
Make changes, commit and push changes
Submit a pull request
Submit an issue
Storage of Jupyter notebooks to explore data
Useful commands
References
Jupyter Notebook and Python for data science.
So, what
is
a Jupyter notebook?
Cool! How do I install Jupyter.
Ok, I’m set! What’s next?
The Future of Jupyter
Reading material
Publicly available databases
Finding data of interest
NCBI
Downloading data from NCBI
Other Protein data
Genomes & Genome Browsers
Other databases full of many things
BLAST
Pathways
Metagenomes
Marine organism resources
Human Genomes
Tool Aggregators
Blogs and other useful links
Downloading and Transferring Files
Download file from the internet to your remote machine
Transfer Files From Your Instance with Filezilla
BWA and samtools and variant calling
Getting started
Download data
Map data
Visualize mapping
Call variants!
Look at the VCF file
Look at the VCF file with bedtools.
Extract reads with samtools.
Discussion points / extra things to cover
Assessing and Assembling Nanopore data
Start a Jetstream instance and install software:
Get Oxford Nanopore MinION data and convert it
Assess the Data
Assemble the data
All-by-all comparisons
Annotate with prokka:
References:
Acknowledgements
canu.report stats from Fundulus olivaceus reads
A sourmash tutorial
At the beginning
K-mers, k-mer specificity, and comparing samples with k-mer Jaccard distance.
K-mers!
K-mers and assembly graphs
Why k-mers, though? Why not just work with the full read sequences?
Long k-mers are species specific
Using k-mers to compare samples against each other
Installing sourmash
Generate a signature for Illumina reads
Compare reads to assemblies
Make and search a database quickly.
Compare many signatures and build a tree.
What’s in my metagenome?
Final thoughts on sourmash
Further Reading:
Exploratory RNAseq data analysis using RMarkdown
Getting started
Make sure R & RStudio are installed:
Download the data for today’s tutorial
Introduction to RMarkdown
Markdown
RMarkdown
Creating a
.Rmd
File
Anatomy of Rmarkdown file
Chunk Labels
Chunk Options
Global Chunk Options
Tables
Citations and Bibliography
Bibliography
Placement
Citation Styles
Citations
Publishing on RPubs
Updating RPubs
Exploratory data analysis with Yeast RNAseq data
Amazing Resources for learning Rmarkdown
RNAseq expression analysis
Make sure R & RStudo are installed:
Install edgeR
Install salmon:
Change to the appropriate directory:
Download some data
Download the yeast reference transcriptome:
Index the yeast transcriptome:
Run salmon on all the samples:
Collect all of the sample counts
Run edgeR (in R)
Extra plotting in R
Questions to ask/address
More reading
Command-line and RStudio
For instructors!
Tutorial authoring guide
E-mail lists
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