RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

Pre-alignment QC


RNA-seq_Flowchart


FastQC

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:

Exercise: Investigate the source/explanation for over-represented sequences:


Fastp

Fastp is a similar alternative tool. QC results for this tool can be produced as follows

cd $RNA_HOME/data
mkdir fastp
cd fastp
wget http://opengene.org/fastp/fastp
chmod a+x ./fastp

mkdir HBR_Rep1 HBR_Rep2 HBR_Rep3 UHR_Rep1 UHR_Rep2 UHR_Rep3
cd $RNA_HOME/data/fastp/HBR_Rep1
../fastp -i $RNA_HOME/data/HBR_Rep1_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/HBR_Rep1_ERCC-Mix2_Build37-ErccTranscripts-chr22.read2.fastq.gz

cd $RNA_HOME/data/fastp/HBR_Rep2
../fastp -i $RNA_HOME/data/HBR_Rep2_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/HBR_Rep2_ERCC-Mix2_Build37-ErccTranscripts-chr22.read2.fastq.gz

cd $RNA_HOME/data/fastp/HBR_Rep3
../fastp -i $RNA_HOME/data/HBR_Rep3_ERCC-Mix2_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/HBR_Rep3_ERCC-Mix2_Build37-ErccTranscripts-chr22.read2.fastq.gz

cd $RNA_HOME/data/fastp/UHR_Rep1
../fastp -i $RNA_HOME/data/UHR_Rep1_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/UHR_Rep1_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz

cd $RNA_HOME/data/fastp/UHR_Rep2
../fastp -i $RNA_HOME/data/UHR_Rep2_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/UHR_Rep2_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz

cd $RNA_HOME/data/fastp/UHR_Rep3
../fastp -i $RNA_HOME/data/UHR_Rep3_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz -I $RNA_HOME/data/UHR_Rep3_ERCC-Mix1_Build37-ErccTranscripts-chr22.read1.fastq.gz


MultiQC

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

cd $RNA_HOME/data
multiqc .

PRACTICAL EXERCISE 4

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

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

Questions

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