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      Advanced Single Cell RNA-Seq Workshop

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Introduction and Lectures
Intro to the Workshop and Core
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What is Bioinformatics/Genomics?
Experimental Design and Cost Estimation
Single Cell Sample Preparation - Dr. Diana Burkart-Waco
Support
Cheat Sheets
Software and Links
Scripts
Prerequisites
CLI - Logging in and Transferring Files
CLI - Intro to Command-Line
CLI - Advanced Command-Line (extra)
CLI - Running jobs on the Cluster and using modules
R - Getting Started
R - Intro to R
R - Prepare Data in R (extra)
R - Data in R (extra)
More Materials (extra)
Data Reduction
Generating Expression Matrices
Expression project setup
Preprocessing reads with HTStream
Generating Expression Tables
VDJ T cell and B cell
Velocity analysis
Data analysis
scRNA analysis prepare
Mapping Comparison
Anchoring (Comparison dataset)
Shiny App Install/Overview
App Practical Usage
AWS Hosted App (Optional)
Monocle
VDJ T cell and B cell analysis
Velocity analysis
ETC
Closing thoughts
Workshop Photos
Github page
Biocore website

Starting a Shiny Server Instance from the Preconfigured Image

Keith Mitchell (kgmitchell@ucdavis.edu)


A few general notes:

Setting up an AWS account for yourself

How to prepare the instance for a client:

  1. Go to the AWS AMI’s (Amazon Machine Images). Select the Rshiny Server Image. Go to Actions>Launch
  2. Next Choose and Instance Type. This will vary in general based on the size of the data you are attempting to analyze. In general it is advised to start with a smaller instance and then move up from there if necessary.
  3. Next Configure the Instance Details. The main step here is to set the “Auto-assign Public IP” to enable.
  4. Keep clicking the bottom right button, “Next: Configure Security Group” until you get to “Step 6: Configure Security Group”
  5. At step 6, select the Rshiny App security group.
  6. Finally launch the instance. (Bottom right hand quarter)
  7. Select an existing key pair or create a new key pair is the final step for starting the instance. Choose “Create a new key pair” and give the key a useful name, here we will use “tutorial”.
  8. Move the downloaded key to ~/.ssh/ via mv ~/Downloads/tutorial.pem ~/.ssh/
  9. chmod 700 ~/.ssh/tutorial.pem
  10. Get the connection to the instance seen highlighted below “ec2-…….amazonaws.com”
  11. Run the following command to upload the “experiment_merged.Rdata” object to the app:
    • scp -i ~/.ssh/tutorial.pem ec2-54-219-166-77.us-west-1.compute.amazonaws.com:/srv/shiny-server/scRNA_shiny_app/ ~/Desktop/experiment_merged.rds
  12. Replace the green section with your Public IV4 DNS address from the instance that was created and used in the previous step.
    • http://ec2-54-219-166-77.us-west-1.compute.amazonaws.com:3838/scRNA_shiny_app/
  13. If there are problems with this link do the following:
    • SSH into the server.
      • ssh -i ~/.ssh/tutorial.key ec2-54-219-166-77.us-west-1.compute.amazonaws.com
    • Restart the shiny service.
      • sudo systemctl restart shiny-server.service
  14. Memory issues?
    • The default memory limit for R on the instance has caused me some problems in the app and it can be tricky to debug.
    • The following link was a solution for this issue but feel free to reach out if you have any other issues in regards to this.
      • https://stackoverflow.com/questions/1395229/increasing-or-decreasing-the-memory-available-to-r-processes
      • You can increase the default using this command, memory.limit(size=2500), where the size is in MB.
      • Or you can check the amount of available memory using memory.limit()