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Organize Kallisto Input Data

Description:

Kallisto has relatively few input requirements. You will need to have either single or paired end reads, as well as a reference transcriptome. It is suggested that your RNA-Seq reads are analyzed using FastQC, followed by any additional trimming and filtering using and application such as Trimmomatic.

Note

About the Sample Dataset In this tutorial, we are using publicly available data from the SRA. This tutorial will start with cleaned and processed reads. The SRA experiment used data from bioproject PRJNA272719. The abstract from that project is reprinted here:

‘To survey transcriptome changes by the mutations of a DNA demethylase ROS1 responding to a phytohormone abscisic acid, we performed the Next-gen sequencing (NGS) associated RNA-seq analysis. Two ROS1 knockout lines (ros1-3, ros1-4; Penterman et al. 2007 [PMID: 17409185]) with the wild-type Col line (wt) were subjected. Overall design: Three samples (ros1-3, ros1-4 and wt), biological triplicates, ABA or mock treatment, using Illumina HiSeq 2500 system’ citation.


Input Data:

Input Description Example
Reference transcriptome fasta Example transcriptome

Importing Reference Transcriptome

In this example, we will import a reference transcriptome for Arabidopsis from Ensembl. In many cases you can find an appropriate transcriptome from Ensembl for your organism of interest, or provide your own fasta-formmatted transcriptome.

  1. Go to the Ensembl Plants Arabidopsis page:http://plants.ensembl.org/Arabidopsis_thaliana/Info/Index.
  2. In the ‘Gene annotation’ section, click on the ‘Download genes, cDNAs, ncRNA, proteins’ ‘FASTA’ link:ftp://ftp.ensemblgenomes.org/pub/plants/release-36/fasta/arabidopsis_thaliana/.
  3. The transcriptome files will be located in the ‘cdna’ folder: ftp://ftp.ensemblgenomes.org/pub/plants/release-36/fasta/arabidopsis_thaliana/cdna/.
  4. Right-click/command-click on the Arabidopsis_thaliana.TAIR10.cdna.all.fa.gz and copy the link location/URL to the clipboard
  5. Login to the CyVerse Discovery Environment.
  6. In your home directory, create a folder to organize your Kallisto project
  7. In the created folder, go to the ‘Upload’ menu, and select, ‘Import from URL…’; paste in the Ensembl link and click ‘Import from URL’ to begin the import

Output/Results

Output Description Example
Reference transcriptome fasta Example transcriptome

Description of results and next steps

This example transcriptome will be indexed in the next step. RNA-Seq reads will be mapped against this set of transcripts. Once you have the transcriptome and your RNA-Seq reads, you can proceed with the next step. We suggest organizing your RNA-Seq reads in the folder created in step 6 above.


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