Pre-alignment QC

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RNA-seq_Flowchart

FastQC

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

http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

Video Tutorial here:

http://www.youtube.com/watch?v=bz93ReOv87Y

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_PUBLIC_IPv4_ADDRESS/rnaseq/data/
Note, you must replace YOUR_PUBLIC_IPv4_ADDRESS with your own amazon instance DNS (e.g., ec2-54-187-159-113.us-west-2.compute.amazonaws.com))
Click on any of the *_fastqc.html files to view the FastQC report

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

HINT: Try BLASTing them.

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
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 ./

Then, go to the following url in your browser:

http://YOUR_PUBLIC_IPv4_ADDRESS/rnaseq/data/multiqc_report.html

Clean up

Move all the FASTQC files into their own directory

cd $RNA_HOME/data
mkdir fastqc
mv *_fastqc* fastqc

PRACTICAL EXERCISE 4

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.

Questions

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.

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