The applications listed here are available for use in the Discovery Environment and are documented in: Discovery Environment Manual.

Discovery Environment Applications List

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  1. Reference Genome (FASTA) or Hisat2 Indexed Reference Genome (in a subdirectory)
  2. Reference Transcriptome (GFF3/GTF/GFF)
  3. RNA-Seq reads (FASTQ) - Single end or Paired-end (compressed or uncompressed) or NCBI SRA id or multiple NCBI SRA id's (each SRA ID on a separate row in the text file).


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Pre-Requisites:

  1. A CyVerse account (Register for a CyVerse account at https://user.cyverse.org). 

  2. An up-to-date Java-enabled web browser. (Firefox recommended. If you wish to work with your own large datasets and upload them using iCommands, Chrome is not suitable due to its issues in utilizing 64-bit Java.)

  3. Mandatory arguments 

    1. Hisat2 reference genomegenome 

      Warningnote

      Select at least one of the below two options for the indexing of the Reference Genome

      1. Custom Reference genome
      2. Hisat2 Indexed folder (Preferred type)
    2. Hisat2 reference annotation

      1. Custom Reference annotation

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      Note

      Use only Only one of the following three options for reads

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      read options has to be selected.

    3. Paired-end reads

      1. FASTQ Files (Read 1): HT path list of read 1 files of paired-end data
      2. FASTQ Files (Read 2): HT path list of read 2 files of paired-end data
    4. Single-end reads

      1. Single end FASTQ files: HT path list of read files of single-end data
    5. SRA

      1. File containing SRA id's: HT path list of multiple SRA ids list files
    6. Cufflinks/Stringtie:    

      Note

      Only one of the below two options needs to be checked.

      Cannot select bothCuffmerge: Run Cuffmerge for Stringtie/Cufflinks 

       Cannot select both

      1. StringTie
      2. Cufflinks
      3. Coverage cut-off threshold: Select from 0-5 (Default is 2)
      4. FPKM cut-off threshold: FPKM cut-off you want to use to filter the transcripts (Default is 2)
    7. Cuffmerge: For merging gtf files produced by Cufflinks/Stringtie (Only works with more than one sample files)
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  4. Advanced options
    1. Phred quality score: encoding for quality score: Phread64 (Default is Phred 33)
    2. Fragment Library Type: specify the format of the library either FR, RF, F, R etc.
    3. Trim bases from 5' end of read: Trim bases from 5' (left) end of each read before alignment (Default is 0)
    4. Trim bases from 3' end of read: Trim bases from 3' (right) end of each read before alignment (Default is 0)

    5. Minimum intron length: Set minimum intron length (Default is 20)
    6. maximum intron length: Set maximum intron length (Default is 500000)
  5. Output
    1. Name of the output folder (Default is Output)

Test/sample data

The following test data are provided for testing RMTA in here - /iplant/home/shared/iplantcollaborative/example_data/OSG-RMTA

  1. Reference Genome: Sorghum_bicolor.Sorbi1.20.dna.toplevel_chr8.fa
  2. Reference Annotation: Sorghum_bicolor.Sorbi1.20_chr8.gtf
  3. left_reads- sample_1_R1.fq.gz
  4. right_reads-sample_1_R2.fq.gz
  5.  Stringtie
  6. Fragment Library Type: FR

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