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. Mandatory fields

    1. Analysis Name

      1. Choose an appropriate name for your analysis and make comments if you wish. Default name is shown in the figure below.

      2. Select the output folder for the results of the analysis.

    2. Genome guided mapping

      Note

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

      1. Custom genome (required)
      2. HISAT2 Indexed folder (for indexed genomes)



    3. Select an aligner

      Note

      ***Only one of the below two options needs to be selected. Both cannot be selected.***

      i) Hisat2
      ii) Bowtie2

      Info
      titleWhen to use HISAT2 or Bowtie2

      HISAT2 is a splice-aware algorithm used to perform reference genome-based read mapping. Stringtie is then used to assemble transcripts based on this read mapping.

      The read aligner Bowtie2 has been included as an optional aligner in the RMTA workflow for users wishing to call single nucleotide polymorphisms (SNPs) from their RNA-seq (or DNA-seq) data in a high throughput manner. When the Bowtie2 option is selected, HISAT2 and Stringtie are both removed from the workflow, but the additional option to remove duplicate reads (important for population level analyses) becomes available.

    4. Reference annotation

      Note

      ***Providing reference annotation is optional and not required when doing de novo transcriptome assembly***

    5. Feature Count Options
      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. Please refer to your Genome Annotation File (.gtf), and confirm that these settings match your data. For Gene Attribute, be sure that gene_id is written before the name of each gene.

    6. Input reads

      Note

      ***Only one of the following three read options (d, e, or f) may be selected per job.***

      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

       

      Note

      ***When inserting multiple paired end FASTQ files, be sure that reads 1 and 2 have matching patterns***

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    1.  

       Single-end reads

                i. Single end FASTQ files or a HT path list of read files of single-end data
                                       Image Modified

         SRA

                i. Enter the SRA id, or

               ii. Select a file containing a list of SRA ids (one per line) or a HT path list of multiple SRA ids list files
                                        Image Modified

       

 

 

 


    1. If you have many files to process through the Discovery Environment, an HT Analysis Path List File may prove useful, as this app takes only 1 file at a time. For information on how to create an HT path list, click here

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    1.  

 

    1. Parameters

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      1. Type of Sequence: Choose either Single End or Paired End

      2. Choose RNA strandedness (default is unstranded)  

      3. Number of threads (Default is 4)

  1.  

      1. Run FastQC

                              Image Added


    1. Advanced options:

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    1.     

      Image Modified

      Info
      titlePhred64, Fastqc, and Remove Duplicate reads options

      Phred gives a quality score of how confidently nucleotides were identified during sequencing. Here, Phred33 is default. Phred 64 should be used if sequencing was performed using Illumina technology 1.3 - 1.8. An error will occur and the run will fail if the wrong quality score has been selected.

      FastQC provides the user with both an overview of potential issues with the data, as well as summary graphs highlighting issues such as per base sequence quality and Kmer content. When the FastQC option has been selected, BAM files are converted back into FASTq, with mapped and unmapped reads, along with their associated quality score retained. This FASTq file is then used as input for FastQC. If issues are detected at the 5’ or 3’ of sequencing reads, RMTA includes additional options for specifically trimming bases off of either end during the next analysis. Sequencing reads of overall poor quality will simply not be mapped and therefore do not need to be trimmed. FASTq files are removed following FastQC analysis.

      If the user chose Bowtie2 as the read aligner and “remove duplicate reads” as an additional option, then the RMTA_Output folder will only contain a sorted BAM file with duplicates removed for each SRA/FASTq input file, as well as a mapped.txt file. No additional files will be generated.

      Info
      titleWhen using Bowtie2

      When using Bowtie2, be sure to check the box labeled "Remove duplicate reads," as shown in the figure below. Duplicate removal is suggested when performing SNP analysis

 



    1. RMTA_Output:

      Name of the output folder (Default is RMTA_Output)
      Image Modified

 



Example Runs:

 

The following test data using Arabidopsis are provided for testing RMTA in here - /iplant/home/shared/iplantcollaborative/example_data/RMTA (this path can be copied and pasted into the navigation bar in a data window within the DE)

 

***Note that when testing SRA IDs, only one of steps 3, 4, or 5 may be used at a time per test run.***  

 

  1. Reference Genome: genome_chr1.fa
  2. Reference Annotation: genome_chr1.gtf 
  3. Paired End Reads: 
    1. Left End Reads: SRR2037320_R1.fastq.gz and SRR2932454_R1.fast.gz
    2. Right End Reads: SRR2037320_R2.fastq.gz and SRR2932454_R2.fastq.gz
  4. Single End Reads: SRR3464102.fastq.gz and SRR3464103.fastq.gz
  5. List of SRA IDs:
    1. Paired End: sra_id_pe.txt
    2. Single End: sra_id_se.txt
  6. Aligners: HISAT2
  7. Feature Count: leave as default
  8. Advanced Options:
    1. Type of Sequence: select Single for Single End and Paired for Paired End 
    2. Leave the rest as default
  9. RMTA Output: leave as default default

 

All other settings should be left as default.

...

Results

Results 

 

Successful execution of RMTA will generate three 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 as well as the Feature_counts and FASTqc (optional) folders.
    1. In turn, this folder will consist of five files associated with each SRA or FASTq:
      1. A filtered GTF (if either the read/base or FPKM filter was set).
      2. A sorted.bam file of all mapped and unmapped reads.
      3. An index associated with the above BAM file.
      4. a GTF that represents the unprocessed file straight out of Stringtie if the user would like to investigate their data further.
      5. A combined.gtf that is the output of the cuffmerge step of RMTA (comparing all identified transcripts back to the reference to identify novel transcripts). This file is useful for novel transcript identification.
    2. The Feature_counts folder contains one file with all of the read count data for each gene across all SRA/FASTq files examined in the run.
    3. The FASTqc_out folder contains subfolders associated with each SRA/FASTq input file. In each folder is an html file with all of the details from the FASTqc run for each set of reads (1 for SE, 2 for PE).
  3. Logfiles: This folder consists of stout and sterr (information written to standard out or standard error log files) files as well as logs specific to running within the DE

 

...

Transcriptome-guided Mapping:

 

Input data requirements:

 

  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 multiple NCBI SRA id's (each SRA ID on a separate row in the text file).

 

Mandatory fields






 
  1. Note

    ***Only one of the following three read options (d, e, or f) may be selected per job.***

  2. 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

      Note

      ***When inserting multiple paired end FASTQ files, be sure that reads 1 and 2 have matching patterns***

  3. Single-end reads

    1. Single end FASTQ files or a HT path list of read files of single-end data


  4. SRA

    1. Enter the SRA id, or
    2. Select a file containing a list of SRA ids (one per line) or a HT path list of multiple SRA ids list files
 

Parameters:

 


 
    1. Type of Sequence: Choose either Single End or Paired End
    2. Choose RNA strandedness (default is unstranded)
    3. Number of threads (Default is 4) 
    4. Run FastQC
 


 

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