Goal

To generate QC reports on Illumina data using FastQC.

Background

In order to inspect the performance of the specific sequencing run, as well as assessing the quality of each sample, quality reports must be generated.

Practical

In this exercise we are going to use the FastQC command line software to generate QC reports for quality assessment. We will run the analysis on all illumina fastq files (if running with 2 cores it should take ~3 minutes).

Instructions

We assume you have FastQC preinstalled prior to this exercise, if not, use conda to install fastqc in a separate environment and load that environment. If conda is not set up, please navigate to https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ and follow instructions for installing FastQC.

We will run the software on all illumina files, if using 2 cores it should take ~ 3 minutes.

1. Before we generate any reports, lets inspect which options are available to use. Execute the software followed by the --help argument.

fastqc --help

• Which arguments would you think could be relevant for this course?

2. Next lets do a minimalistic run and see what happens, lets be lasy and point to all files quickly. In the terminal, navigate to the folder containing all Illumina files and run.

mkdir fastqc_reports
fastqc --outdir fastqc_reports --threads 2 Ec00*.illumina*fastq.gz

Here we use wildcards to specify which patterns the filenames should have, we do this to ensure that if any nanopore files are stored in the same directory, only the Illumina files will be selected. Additionally, we utilize the --outdir flag to ensure that results are redirected to a folder we dersire.

3. FastQC produced several files as output, most importantly it produces one QC report (HTML file) per input file.

• How many reports would we expect for each sample?

• What other type of file(s) do FastQC produce?
  • What are their contents?

4. Congratz, you just made your first Illumina QC reports 😀