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LEARNING MATERIALS
 

 

 

 

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Rationale and background:

Overview of rnaQUAST

rnaQUAST is a tool for evaluating RNA-Seq assemblies using reference genome and gene data database. In addition, rnaQUAST is also capable of estimating gene database coverage by raw reads and de novo quality assessment using third-party softwares (STAR, TopHat, GMAP etc.,). The detailed manual is available here - http://spades.bioinf.spbau.ru/rnaquast/release1.1.0/manual.html

Needs to run rnaQUAST on Atmosphere

  1. Atmosphere requirements
    1. CyVerse username that has an institutional email (e.g. janedoe@email.arizona.edu)
    2. Atmosphere allocation
      1. Follow section called "Adding apps and services to your account"
      2. Fill in the request sheet with the allocation needed
        1. Include the number of Atmosphere Units (AUs) that you will need
        2. It can be difficult to know how AUs many are needed a priori,
          1. (number of cores) x (real time hours) x (days you need to run the instance) = AUs needed in a month
  2. Computational Knowledge
    1. Familiarity with the terminal/shell

Learn about allocations

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Learn about CyVerse's allocation policies here.

Protocol: How to use rnaQUAST 1.1.0 on Atmosphere?

This tutorial will take users through the steps of:

  1. Launching the rnaQUAST 1.1.0 Atmosphere image
  2. Running rnaQUAST 1.1.0 on a test data 

Please work through the tutorial and add your comments on the bottom of this page. Or send comments per email to upendra@cyverse.org. Thank you

Part 1: Connect to an instance of an Atmosphere Image (Virtual Machine)

Step 1. Go to https://atmo.iplantcollaborative.org and log in with your CyVerse credentials.

Step 2. Click on the Launch New Instance button and search for rnaQUAST 1.1.0.

Step 3. Select the image rnaQUAST 1.1.0 and click Launch Instance. It will take 10-15 minutes for the cloud instance to be launched. 

Note: Instances can be configured for different amounts of CPU, memory, and storage depending on user needs.  This tutorial can be accomplished with the medium instance size, medium1 (4 CPUs, 8 GB memory, 80 GB root)  

Part 2: Set up a rnaQUAST 1.1.0 run on a test data using the Terminal window

Step 1. Open the Terminal.  Add the ssh details along with your IP address to connect the instance through the terminal

step 2. You will find rnaQUAST v1.1.0 software in "/opt" folder. All the dependencies for running rnaQUAST v1.1.0 are located in "/opt/rnaQUAST-1.1.0"  

Step 3Before you start using the rnaQUAST 1.1.0, you need to make sure that the following softwares are added to your PATH

Step 4. The staged example data can be found in folder "rnaQUAST-1.1.0/test_data" within "opt" folder.  List its contents with the ls command:

  • Trinity.fasta, spades.311.fasta and idba.fasta are test assemblies assembled with Trinity, spades and idba respectively
  • Saccharomyces_cerevisiae.R64-1-1.75.gtf is the test reference annotation file
  • Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.fa is the test reference genome file. The files with extension (.bt2) are bowtie2 indexed reference genome files
  • Paired_end1.fq and Paired_end2.fq are the test paired end fastq read files
 
Step 5. Set up a rnaQUAST 1.1.0 test run to check that everything is installed correctly and all the software are added to the $PATH environmental variable

 

Basic run:

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It is recommended to redirect the output to home directory because of the disk space restrictions on /opt directory

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By default, the rnaQUAST 1.1.0 uses GMAP. If you want to run the above test using BLAT option.

 

a. Using rnaQUAST 1.1.0 tool using GMAP  (default)

b. Using rnaQUAST 1.1.0 tool using BLAT

Read alignment:

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rnaQUAST 1.1.0 is also capable of calculating various statistics using raw reads (e.g. database coverage by reads) using either STAR aligner (or alternatively TopHat aligner +SAM tools)

a. Using rnaQUAST 1.1.0 tool using STAR aligner (default)

 b. Using rnaQUAST 1.1.0 tool using TopHat aligner +SAM tools

Software for de novo quality assessment:

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When reference genome and gene database are unavailable, it is recommend to run BUSCO and GeneMarkS-T in rnaQUAST tool.

a. Using rnaQUAST 1.1.0 tool with BUSCO 

 b. Using rnaQUAST 1.1.0 tool with GeneMarkS-T 

Outputs

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For detailed explanation of the outputs with each of the above runs, please refer to rnaQUAST 1.1.0 manual

 


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