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

Discovery Environment Applications List

The box below searches only this space.
To search the entire iPlant wiki, enter your query in the box at the upper right.

 

 

 

 

Versions Compared

Key

  • This line was added.
  • This line was removed.
  • Formatting was changed.

...

RMTA is a wrapper script built on top of several publicly available bioinformatic tools that can rapidly proceed from raw short read data to assembled transcripts. RMTA performs this by mapping reads using either HiSat2 or Bowtie2 and then assembling transcripts using either Cufflinks or StringTie, according to user preference. RMTA can process FASTQ files containing paired-end or single-end reads or can directly process one or more sequence read archives (SRA) from NCBI using SRA IDs. RMTA has been successfully used by many groups as the first step towards the identification of long non-coding RNAs using the Evolinc workflow. More information about RMTA can be found here

...


Pre-Requisites:

  1. A CyVerse account (Register for a free 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. Reference genome 

      Note

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

      1. Custom genome
      2. Reference genome from the list
      3. Hisat2 Indexed folder (Preferred type)
    2. Reference annotation

      1. Custom Reference annotation
      2. Reference Annotation from the list

      Note

      Only one of the following three 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. Enter the SRA id
      2. Select a file containing SRA ids: HT path list of multiple SRA ids list files
    6. Aligners

      Note

      Only one of the below two options needs to be selected. Cannot select both Both cannot be selected.

      1. Hisat2
      2. Bowtie2

    7. Featurecount
      1. Choose a Feature Type. The default option will be "exon"
      2. Choose a gene attribute. The default option will be "gene_id"
      3. Select the type of strandedness. The three options include unstranded, stranded, and reversely stranded.

  4. Advanced options
    1. Type of Sequence: Choose either Single End or Paired End
    2. Number of threads (Default is 4)
    3. FPKM cut-off threshold (For RNA-Seq reads only with Hisat2) (Default is 0)
    4. Coverage cut-off threshold (For RNA-Seq reads only with Hisat2) (Default is 0)
    5. Choose RNA strandedness (default is unstranded) 
    6. Trim bases from 5' end of read: Trim bases from 5' (left) end of each read before alignment (Default is 0)
    7. Trim bases from 3' end of read: Trim bases from 3' (right) end of each read before alignment (Default is 0)
    8. Minimum intron length: Set minimum intron length (Default is 20)
    9. Maximum intron length: Set maximum intron length (Default is 500000)
    10. Phred64 (Default is Phred33): Check to run Phred64
    11. Run Fastqc
    12. Remove duplicate reads
  5. RMTA_Output
    1. Name of the output folder (Default is RMTA_Output)
  6. README
    1. HISAT2 and BOWTIE2 are fast and sensitive alignment program for mapping next-generation sequencing reads (both DNA and RNA). StringTie is a fast and highly efficient assembler of RNA-Seq alignments into potential transcripts. Cuffcompare compare compares your assembled transcripts to a reference annotation and track tracks Cufflinks transcripts across multiple experiments (e.g. across a time course).

Example Runs

...

  1. Reference Genome: genome_chr1.fa
  2. Reference Annotation: genome_chr1.gtf
  3. left_reads- sample_1_R1.fq.gz
  4. right_reads-sample_1_R2.fq.gz
  5. Stringtie
  6. Fragment Library Type: FR

Leave the rest All other settings should be left as default.

Results 

Successful execution of RMTA will generate two output folders:

  1. Index: This folder consists of the index of the genome
  2. Output: This folder consists of the output from Hisat2, Stringtie and Cuffcompare (Please refer to the manual for the explanation of outputs from these individual programs)