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

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
Computational Knowledge
1. Familiarity with the terminal/shell

Learn about allocations

Learn about CyVerse's allocation policies here.

Protocol: How to use rnaQUAST 1.2.0 on Atmosphere?

This tutorial will take users through the steps of:

Launching the rnaQUAST 1.2.0 Atmosphere image
Running rnaQUAST 1.2.0 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 1.2.0.

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

$ ssh <username>@Ipaddress

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"

$ cd /opt/rnaQUAST-1.1.0
$ ls rnaQUAST-1.1.0
augustus-3.2.1  download     gmap-2015-12-31                 initial.meta.lst.feature  manual.html        other_libs  rnaQUAST.py           seq_bin_3                  VERSION
blat            final_model  gms.log                         itr_0.lst                 metrics            quast23     rnaQUAST_test_output  STAR-2.5.1b
bowtie2-2.2.7   GeneMarkS-T  GPLv2.txt                       LICENSE                   ncbi-blast-2.3.0+  README      seq_bin_1             test_data
BUSCO_v1.1b1    general      hmmer-3.1b2-linux-intel-x86_64  Log.out                   objects            report      seq_bin_2             tophat-2.1.0.Linux_x86_64

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

export PATH=/opt/rnaQUAST-1.1.0/ncbi-blast-2.3.0+/bin/:$PATH
export PATH=/opt/rnaQUAST-1.1.0/blat/:$PATH
export PATH=/opt/rnaQUAST-1.1.0/BUSCO_v1.1b1/:$PATH
export PATH=/opt/rnaQUAST-1.1.0/GeneMarkS-T/:$PATH
export PATH=/opt/rnaQUAST-1.1.0/STAR-2.5.1b/bin/Linux_x86_64_static/:$PATH
export PATH=/opt/rnaQUAST-1.1.0/tophat-2.1.0.Linux_x86_64/:$PATH
export PATH=/opt/rnaQUAST-1.1.0/bowtie2-2.2.7/:$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:

$ cd /opt/rnaQUAST-1.1.0
$ ls test_data
checkpoint       Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.1.bt2  Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.fa         spades.311.fasta
idba.fasta       Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.2.bt2  Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.rev.1.bt2  Trinity.fasta
Paired_ends1.fq  Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.3.bt2  Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.rev.2.bt2
Paired_ends2.fq  Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.4.bt2  Saccharomyces_cerevisiae.R64-1-1.75.gtf

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

$ python rnaQUAST.py --test

Basic run:

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

a. Using rnaQUAST 1.1.0 tool using GMAP (default)

$ python rnaQUAST.py --threads 4 --transcripts test_data/idba.fasta test_data/spades.311.fasta test_data/Trinity.fasta --reference test_data/Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.fa --gene_database test_data/Saccharomyces_cerevisiae.R64-1-1.75.gtf --output_dir ~/rnaQUAST_test_output_GMAP

b. Using rnaQUAST 1.1.0 tool using BLAT

$ python rnaQUAST.py --threads 4 --blat --transcripts test_data/idba.fasta test_data/spades.311.fasta test_data/Trinity.fasta --reference test_data/Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.fa --gene_database test_data/Saccharomyces_cerevisiae.R64-1-1.75.gtf --output_dir ~/rnaQUAST_test_output_BLAT

Read alignment:

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)

$ python rnaQUAST.py --threads 2 --transcripts test_data/idba.fasta test_data/spades.311.fasta test_data/Trinity.fasta --left_reads test_data/Paired_ends1.fq --right_reads test_data/Paired_ends2.fq --reference test_data/Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.fa --gene_database test_data/Saccharomyces_cerevisiae.R64-1-1.75.gtf --output_dir ~/rnaQUAST_test_output_STAR

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

$ python rnaQUAST.py --no_plots --threads 2 --transcripts test_data/idba.fasta test_data/spades.311.fasta test_data/Trinity.fasta --left_reads test_data/Paired_ends1.fq --right_reads test_data/Paired_ends2.fq --reference test_data/Saccharomyces_cerevisiae.R64-1-1.75.dna.toplevel.fa --gene_database test_data/Saccharomyces_cerevisiae.R64-1-1.75.gtf --output_dir ~/rnaQUAST_test_output_TOPHAT --tophat

Software for de novo quality assessment:

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

$ python rnaQUAST.py --threads 4 --transcripts test_data/idba.fasta test_data/spades.311.fasta test_data/Trinity.fasta --output_dir ~/rnaQUAST_test_output_arthropoda_BUSCO --disable_infer_genes --disable_infer_transcripts --busco --clade /opt/rnaQUAST-1.1.0/BUSCO_v1.1b1/arthropoda

b. Using rnaQUAST 1.1.0 tool with GeneMarkS-T

$ python rnaQUAST.py --threads 4 --transcripts test_data/idba.fasta test_data/spades.311.fasta test_data/Trinity.fasta --output_dir ~/rnaQUAST_test_output_GM --gene_mark

Outputs

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

rnaQUAST 1.2.0 using Atmosphere