rnaQUAST 1.1.0 using Atmosphere
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
- CyVerse username that has an institutional email (e.g. janedoe@email.arizona.edu)
- Atmosphere allocation
- Follow section called "Adding apps and services to your account"
- Fill in the request sheet with the allocation needed
- Include the number of Atmosphere Units (AUs) that you will need
- It can be difficult to know how AUs many are needed a priori,
- (number of cores) x (real time hours) x (days you need to run the instance) = AUs needed in a month
- Computational Knowledge
- Familiarity with the terminal/shell
Learn about allocations
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:
- Launching the rnaQUAST 1.1.0Â Atmosphere image
- Running rnaQUAST 1.1.0Â on a test dataÂ
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.Â
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) Â
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" Â
$ 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. Â 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
$PATH
 environmental variableÂ
$ 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
By default, the rnaQUAST 1.1.0 uses GMAP. If you want to run the above test using BLAT option.
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a. Using rnaQUAST 1.1.0 tool using GMAPÂ Â (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:
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 BUSCOÂ
$ 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-TÂ
$ 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
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