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Table of Contents


Now users can run Rstudio as app in CyVerse Discovery Environment. Please see this updated wiki page to run New RNA-Seq Tuxedo protocol - RNA-seq Tutorial- HISAT2, StringTie and Ballgown using DE and Rstudio-Ballgown*


Alessandra Fracasso,  Luisa M. Trindade and Stefano Amaducci; DOI: 10.1186/s12870-016-0800-x, May 2016

The Staged Fastq Data can be found in the


We will use StringTie-1.3.3 again to assemble transcripts but this time will will use the consolidated annotation file we got from StringTie-1.3.3_merge step. This will re-estimate the transcript abundances using the merged structures but reads may need to be re-allocated for transcripts whose structures were altered by the merging step. We will set the option -B, e  of StringTie which . These options are set by checking the two boxes at the bottom of the 'analysis option' section of the app.  This will create table count files (*ctab files) for each sample to be used in Ballgown for differential expression.Name the analysis "StringTie-1.3.3_from_merged_annotation"


Run the App. When it is complete, you should see the following outputs under


  • Ballgown_input_files: count files (*ctab files) for each sample

    • e_data.ctab: exon-level expression measurements.

    • i_data.ctab: intron- (i.e., junction-) level expression measurements

    • t_data.ctab: transcript-level expression measurements

    • e2t.ctab: table with two columns, e_id and t_id, denoting which exons belong to which transcripts

    • i2t.ctab: table with two columns, i_id and t_id, denoting which introns belong to which transcripts


More details of the ctab files please refere the Ballgown documentation


Section 5: Compare expression analysis using Ballgown


ID      condition   reps

IS20351_DS_1_1    DS     1

IS20351_DS_1_2     DS     2

IS20351_DS_1_3     DS     3

IS20351_WW_1_1    WW     1

IS20351_WW_1_2    WW     2

IS20351_WW_1_3    WW     3

Here we are doing a pairwise comparison for differential expression in sensitive genotype under Drought Stress(DS) and Well Watered(WW) condition. We have 3 replicates under each condition.


4. Once the instance is active, you will get a email confirmation and can also see  the status active on the Atmosphere portal

5. Copy the IP address "", if you are using MAC or Ubuntu open a terminal window and paste the IP address on the command line using your Cyverse user name and hit enter. This will ask you your Cyverse user account passowrd, hit enter
Note- After entering the password if its ask you to add your IP address to the config, enter yes.