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RNA STAR (option for 50 % of attendees)

For information to set proper value for STAR parameters:

  1. create a new history and name it RNA STAR
  2. Import the 11 datasets from the RNAseq data library to this RNA STAR history, plus the Drosophila_melanogaster.BDGP6.95.gtf file
  3. Select the RNA STAR tool with the following parameters to map your reads on the reference genome:
    1. Single-end or paired-end reads: Single-end
    2. RNA-Seq FASTQ/FASTA file (multiple datasets button), Cmd-shift Select:
      • GSM461176_untreat_single.fastq.gz
      • GSM461179_treat_single.fastq.gz
    3. Custom or built-in reference genome: Use a built-in index
    4. Reference genome with or without an annotation: use genome reference without builtin gene-model
    5. Select reference genome: Drosophila Melanogaster (dm6)
    6. Gene model (gff3,gtf) file for splice junctions: the imported Drosophila_melanogaster.BDGP6.95.gtf
    7. Length of the genomic sequence around annotated junctions: 44 (This parameter should be length of reads - 1, see above table from fastQC/multiQC analysis)
  4. Execute

Redo the STAR run with

3.2 Select the RNA STAR tool with the following parameters to map your reads on the reference genome: RNA-Seq FASTQ/FASTA file (as multiple datasets), Cmd-shift Select: - GSM461182_untreat_single.fastq.gz

3.7 Length of the genomic sequence around annotated junctions: 74 (This parameter should be length of reads - 1, see above table from fastQC/multiQC analysis)

Redo a last STAR run for paired-end datasets

  1. With the following parameters to map your reads on the reference genome:
    1. Single-end or paired-end reads: Paired-end (as multiple datasets)
    2. RNA-Seq FASTQ/FASTA file, forward reads (multiple datasets button), Cmd-shift Select:
      • GSM461177_1_untreat_paired.fastq.gz
      • GSM461178_1_untreat_paired.fastq.gz
      • GSM461180_1_treat_paired.fastq.gz
      • `GSM461181_1_treat_paired.fastq.gz
    3. RNA-Seq FASTQ/FASTA file, forward reads (multiple datasets button), Cmd-shift Select:
      • GSM461177_2_untreat_paired.fastq.gz
      • GSM461178_2_untreat_paired.fastq.gz
      • GSM461180_2_treat_paired.fastq.gz
      • `GSM461181_2_treat_paired.fastq.gz
    4. Custom or built-in reference genome: Use a built-in index
    5. Reference genome with or without an annotation: use genome reference without builtin gene-model
    6. Select reference genome: Drosophila Melanogaster (dm6)
    7. Gene model (gff3,gtf) file for splice junctions: the imported Drosophila_melanogaster.BDGP6.95.gtf
    8. Length of the genomic sequence around annotated junctions: 36 (This parameter should be length of reads - 1, see above table from fastQC/multiQC analysis)
  2. Execute

Rename your datasets !

You need now to rename you datasets to facilitate your downstream analysis.

Be quiet and focus ! No hurry, this is an important task in the analysis.

  1. Search and select datasets with RNA STAR

  2. Click on the info icon of both log and bam files

  3. Copy the name or one of the two names of the datasets as shown bellow

  4. Now click on the pencil icon of the same dataset
  5. Paste your text in the Name field of the dataset
  6. Edit your text as follow for log files

  7. Edit your text as follow for bam files

  8. repeat ad lib for all log and bam files