PreAlignment QC

Obi Griffith edited this page May 30, 2018 · 41 revisions

RNA-seq Flowchart - Module 1

1-vi. Pre-Alignment QC

You can use FastQC to get a sense of your data quality before alignment:

Video Tutorial here:

Try to run FastQC on your fastq files:

cd $RNA_HOME/data
fastqc *.fastq.gz

Then, go to the following url in your browser:

  • http://YOUR_DNS_NAME/workspace/rnaseq/data/
  • Note, you must replace YOUR_DNS_NAME with your own amazon instance DNS (e.g.,
  • Click on any of the *_fastqc.html files to view the FastQC report


Investigate the source/explanation for over-represented sequences:

  • HINT: Try BLASTing them.


Assignment: Run FASTQC on one of the additional fastq files you downloaded in the previous practical exercise.

  • Hint: Remember that you stored this data in a separate working directory called ‘practice’.

Run FASTQC on the file 'hcc1395_normal_1.fastq.gz' and answer these questions by examining the output.


  • How many total sequences are there?
  • What is the range (x - y) of read lengths observed?
  • What is the most common average sequence quality score?
  • What does the Adaptor Content warning tell us?

Solution: When you are ready you can check your approach against the Solutions

Run MultiQC on your fastqc reports to generate a single summary report across all samples/replicates.

cd $RNA_HOME/data
multiqc .

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