Remember there is no “one right way” to do an analysis. Choose parameters that you think are the most suitable for your goal.

• Create an account at https://usegalaxy.eu/

• Load following fastq files as a Collection (List of Pairs):

  • https://zenodo.org/record/3541678/files/A1_left.fq.gz
    

  • https://zenodo.org/record/3541678/files/A1_right.fq.gz

  • https://zenodo.org/record/3541678/files/A2_left.fq.gz
  • https://zenodo.org/record/3541678/files/A2_right.fq.gz
  • https://zenodo.org/record/3541678/files/A3_left.fq.gz
  • https://zenodo.org/record/3541678/files/A3_right.fq.gz
  • https://zenodo.org/record/3541678/files/B1_left.fq.gz
  • https://zenodo.org/record/3541678/files/B1_right.fq.gz
  • https://zenodo.org/record/3541678/files/B2_left.fq.gz
  • https://zenodo.org/record/3541678/files/B2_right.fq.gz
  • https://zenodo.org/record/3541678/files/B3_left.fq.gz
  • https://zenodo.org/record/3541678/files/B3_right.fq.gz

• Run FastQC before and after trimming reads with Trimmomatic. Trim for quality and consider whether the adaptor removal should be performed.

• Assemble the trimmed reads with Trinity. Trinity will output both gene and isoform files. Focus on the isoforms.

• Align trimmed reads to this de-novo reference assembly and estimate read abundance per isoform (Align reads and estimate abundance on a de novo assembly of RNA-Seq data). Use salmon as Abundance estimation method.

• Rename the datasets: A1_raw, A2_raw, A3_raw, B1_raw, B2_raw, B3_raw

• Build expression matrix for your de novo assembly of RNA-Seq data by Trinity (this is the first step in the differential gene expression pipeline)

• Share your history with the user cechova.biomonika@gmail.com

• Export your history to a file and upload your .tar.gz to the Odevzdávarna by April 13th, 2021

This exercise is inspired by the following draft tutorial: