- Home
- Experimental Design
- Challenges to DE analysis
- Inputs
- Check sequence quality (You are here)
- Map reads
- Generate expression matrix
- Import data into R
- Screen for outlier samples
- edgeR
- DESeq2
We will use the FastQC program to check for commonly encountered sequence quality issues. This program generates a long series of plots and tables summarizing various aspects of sequence quality for a FASTQ file. Some nice examples of output generated using good datasets as well as datasets with different types of issues are included on the site and you are highly encouraged to go through those materials to get a better sense of how to interpret the output. More detailed descriptions of what each plot represents is found in the web pages listed here.
The basic command we will use for executing fastqc on each input FASTQ file will look like this:
fastqc -t $cpus "$fastq"
where $cpus is the number of vCPUS/threads available and $fastq is the path to the FASTQ file we want to evaluate.
The FastQC program and its dependencies are not available on the host system, like the gunzip command we used in the Inputs chapter. Instead, FastQC is packaged in the singularity container /projects/researchit/crf/containers/crf_rnaseq.sif, so the more complete command will look like this:
container=/projects/researchit/crf/containers/crf_rnaseq.sif
singularity exec "$container" fastqc -t $cpus "$fastq"
In order to facilitate non-interactive, multi-threaded batch execution on the JAX HPC cluster, using reasonable numbers of computational threads and RAM, we have included a slurm convenience script fastqc.sh, which wraps this process and can be invoked as follows:
sbatch fastqc.sh "$fastq"
If we have a directory of files, we can use this script and a bit of bash to process all the FASTQ files in the directory:
## make a separate directory in which to do the work for this step:
mkdir -p /fastscratch/$USER/rnaseq/fastqc
## copy the script over and link the data files:
cd /fastscratch/$USER/rnaseq
cp ~/opt/crf_rnaseq_basics/scripts/fastqc.sh fastqc ## configure path for your install
ln -s $PWD/data/*.fastq fastqc
cd fastqc
$ ls -l
total 0
-rw-r--r-- 1 user15 group12 881 Feb 11 10:00 fastqc.sh
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849386.fastq -> /fastscratch/user15/rnaseq/data/SRR11849386.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849387.fastq -> /fastscratch/user15/rnaseq/data/SRR11849387.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849388.fastq -> /fastscratch/user15/rnaseq/data/SRR11849388.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849389.fastq -> /fastscratch/user15/rnaseq/data/SRR11849389.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849390.fastq -> /fastscratch/user15/rnaseq/data/SRR11849390.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849391.fastq -> /fastscratch/user15/rnaseq/data/SRR11849391.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849392.fastq -> /fastscratch/user15/rnaseq/data/SRR11849392.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849393.fastq -> /fastscratch/user15/rnaseq/data/SRR11849393.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849394.fastq -> /fastscratch/user15/rnaseq/data/SRR11849394.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849395.fastq -> /fastscratch/user15/rnaseq/data/SRR11849395.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849396.fastq -> /fastscratch/user15/rnaseq/data/SRR11849396.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849397.fastq -> /fastscratch/user15/rnaseq/data/SRR11849397.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849398.fastq -> /fastscratch/user15/rnaseq/data/SRR11849398.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849399.fastq -> /fastscratch/user15/rnaseq/data/SRR11849399.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849400.fastq -> /fastscratch/user15/rnaseq/data/SRR11849400.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849401.fastq -> /fastscratch/user15/rnaseq/data/SRR11849401.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849402.fastq -> /fastscratch/user15/rnaseq/data/SRR11849402.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849403.fastq -> /fastscratch/user15/rnaseq/data/SRR11849403.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849404.fastq -> /fastscratch/user15/rnaseq/data/SRR11849404.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849405.fastq -> /fastscratch/user15/rnaseq/data/SRR11849405.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849406.fastq -> /fastscratch/user15/rnaseq/data/SRR11849406.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849407.fastq -> /fastscratch/user15/rnaseq/data/SRR11849407.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849408.fastq -> /fastscratch/user15/rnaseq/data/SRR11849408.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849409.fastq -> /fastscratch/user15/rnaseq/data/SRR11849409.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849410.fastq -> /fastscratch/user15/rnaseq/data/SRR11849410.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849411.fastq -> /fastscratch/user15/rnaseq/data/SRR11849411.fastq
lrwxrwxrwx 1 user15 group12 49 Feb 11 10:00 SRR11849412.fastq -> /fastscratch/user15/rnaseq/data/SRR11849412.fastq
## launch jobs in parallel in the background; print corresponding job_ids:
for f in *.fastq; do
echo -n "$f: "
sbatch fastqc.sh "$f"
done
The main output file of interest will have the same prefix as the FASTQ file (everything up to '.fastq') followed by the extension '_fastqc.html'. This file can be viewed with your web browser and contains all the expected plots.