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featureCounts

  1. In your history HISAT2 or STAR
  2. Select the featureCounts tool with the following parameters to count your reads:
    1. Alignment file: select multiple datasets button and shift-click the 7 bam files you have generated
    2. Specify strand information: Unstranded
    3. Gene annotation file : in your history
      • Gene annotation file: Drosophila_melanogaster.BDGP6.95.gtf
    4. FASTA/Q file: Gene-ID "\t" read-count (MultiQC/DESeq2/edgeR/limma-voom compatible)
    5. Create gene-length file: Yes
    6. In Options for paired-end reads:
      • Count fragments instead of reads: Enabled; fragments (or templates) will be counted instead of reads
    7. In Advanced options:
      • GFF feature type filter: exon
      • GFF gene identifier: gene_id
      • Allow read to contribute to multiple features: No
      • Count multi-mapping reads/fragments: Disabled; multi-mapping reads are excluded (default)
      • Minimum mapping quality per read: 10
    8. Leave other settings as defaults
  3. Execute

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 featurecounts (as we did before for renaming datasets)

  2. Click on the info icon of featureCounts on xxx: Counts

  3. Copy the name of the dataset Alignment file in the tool parameters table (for instance, GSM461176_untreat_single.bam)

  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 by replacing bam by Counts (e.g. GSM461176_untreat_single.Counts)
  7. repeat ad lib for all counts files generated by featureCounts

MultiQC

We have now generated (1) Bam alignments and (2) Counts files with feature counts, and we have carefully and courageously edited the names of generated datasets. We are going to be rewarded for this effort in the next steps !

  1. In your history HISAT2 or STAR
  2. Select the MultiQC tool with the following parameters:
    1. 1: Results/ Which tool was used generate logs?: STAR or HISAT2 (depending on your analysis track)
    2. STAR or HISAT output: shift-click select all files with the extension .log
    3. Click on the + Insert Result button
    4. 2: Results/ Which tool was used generate logs?: featureCounts
    5. Output of FeatureCounts: shift-click select all files with the extension : Summary
  3. Execute

Examine the results and