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Pre-Requisites

1. A CyVerse account. (Register for an CyVerse account here - user.cyverse.org)

2. Before using the Ballgown R package, a few preprocessing steps are necessary:

   1. RNA-Seq reads should be aligned to a reference genome.
   2. A transcriptome should be assembled, or a reference transcriptome should be downloaded.
   3. Expression for the features (transcript, exon, and intron junctions) in the transcriptome should be estimated in a Ballgown readable format.
3. Two sample pipelines for preprocessing are as follows:
   1. Pipeline 1: TopHat2 + Stringtie
   2. Pipeline 2: TopHat2  + Cufflinks  + Tablemaker
   3. Both the above pipelines give Ballgown readable format outputs
4. Mandatory arguments 
   1. Directory of ctab files from stringtie output
   2. design matrix file e.g
      

      ID      group   reps

      IS22330_DS_1_.sorted    tol     1

      IS22330_DS_2_.sorted    tol     2

      IS22330_DS_3_.sorted    tol     3

      IS20351_DS_1_.sorted    sen     1

      IS20351_DS_2_.sorted    sen     2

      IS20351_DS_3_.sorted    sen     3
      
      

   3. covariate of the experiment: covariate of interest e.g case/control, status or time. The name should match the cloumn name in the desgin_matrix file; in this example its "group"