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Quick start of RNA-seq analysis in DE

Goal

Perform RNAseq differential expression analysis using Read Mapping and Transcript Assembly (RMTA) and Rstudio-DESEq2 apps

  • RMTA is a workflow that can rapidly process raw RNA-seq illumina data by mapping reads to a genome (HISAT2), assemble transcripts using Stringtie and count reads using featureCounts.
  • DESeq2 is a R package to analyze count tables for differentially expressed genes, visualize the results, add extra gene annotations, and cluster samples and genes using transformed counts

In this quick start, we will show you how to launch RMTA app and Rstudio-DESeq2 VICE app in DE

Prerequisites

Downloads, access, and services

In order to complete this tutorial you will need access to the following services/software

Prerequisite Preparation/Notes Link/Download
CyVerse account You will need a CyVerse account to complete this exercise Register

Platform(s)

We will use the following CyVerse platform(s):

Platform Interface Link Platform Documentation Learning Center Docs
Discovery Environment Web/Point-and-click Discovery Environment DE Manual Guide

Test data for webinar

RNAseq_Webinar_test_data

Launch RMTA_v2.6.1 with test data

Right-Click the below button and open in a new tab for quick launch of RMTA-2.6.1 analysis with test data

rmta-2.6.1
  1. Login to the Discovery Environment.
  2. CLick on “Apps” tab in the Discovery Enviornment and search for “RMTA v2.6.1”.
  3. Click on the three dots button near the top-right corner of the app and click on “Quick Launch”.

RMTA_quick_launch_1

  1. Click on the “RNAseq_Webinar_RMTA” quick launch and click on the play button to launch.

RMTA_quick_launch_3

  1. Under “Analysis Name” leave the defaults or make any desired notes.

    Note

    The quick launch feature comes pre-loaded with required files (reference genome, annotation and single-end fastq reads) for performing read mapping and transcript assembly of the test data.

  2. Under “Resource Requirements” request resources as needed or leave for defaults

  3. Click Launch Analysis. You will receive a notification that the job has been submitted and running.

Note

You will be notified when the analysis has finished successfully.

8. Click on the “Analyses” button to display the dashboard of your analyses. Click on your RMTA anlaysis to navigate to that analysis folder in your data store.

Note

We will need the “feature_counts.txt” file from the “Feature_counts” folder in the “RMTA_Output” folder for next differential expression analysis.

Launch Rstudio-DESEq2 VICE app with test script

VICE is a Visual and Interactive Computing Environment which is the latest feature in CyVerse’s Discovery Environment (DE) for running interactive apps such as Rstudio and Jupyter Notebooks.

Right-Click the below button and open in a new tab for quick launch of Rstudio-DESeq2 VICE app

RMTA-deseq2
  1. CLick on “Apps” tab in the Discovery Enviornment and search for “Rstudio-DESeq2”.
  2. Click on the three dots button near the top-right corner of the app and click on “Quick Launch”.

DESeq2_quick_launch_1

  1. Click on the “RNAseq_Webinar_DESEq2” quick launch and click on the play button to launch.

DESeq2_quick_launch_3

  1. Under “Analysis Name” leave the defaults or make any desired notes.

  2. Under “Select input files and folder” add the “feature_counts.txt” file we got from our previous RMTA run.

    Note

    The quick launch feature comes pre-loaded with DESeq2 R script.

  3. Under “Resource Requirements” request resources as needed or leave for defaults.

  4. Click Launch Analysis. You will receive a notification that the job has been submitted and running with the “Access your running analysis here”.

  5. Clicking on the “Access your running analysis” will open the Rstudio-DESeq2 in another tab in the browser after a brief building phase.

Note

You will be asked to authenticate again to the Rstudio with your CyVerse username and password

  1. The username and password for logging into Rstudio are “rstudio”.
  2. Finally, once you finish analysis, navigate to the DE tab, select the Analysis window and select the analysis, click “save and complete analysis”. Upon clicking complete analysis, the analysis will be completed and all the outputs will be brought back to the analysis folder.

Note

To use our example script, navigate to Community Data > *iplantcollaborative > example_data > RNAseq_Webinar *

Additional information, help

Fix or improve this documentation

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