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Sequencing: Quality Check by fastqc

Bio-tech

This article to demonstrate how to do quality check for the data from Illumina Sequencer.

Download FASTQ file

Use publicly uploaded sequencing data on DRA (DDBJ Sequence Read Archive) [1]. You can also download same data from SRA (NCBI Sequence Read Archive) or ERA(EBI Sequence Read Archive [1].

Go to DRASearch page, clicking Search button on the top of the page [1].

On the DRASearch page, I selected

  • Organism: Homo sapiens
  • StudyType: Exome Sequencing
  • Platform: Illumina

f:id:happybirthday:20180422192617p:plain

This time, I would select a submitted data Submission ID: SRA067162 [2] whose base number is small (1.3 G bases). Thank you for sharing the data!

You can see FASTQ data file [3][4] links in the right side of [2]. The FASTQ (.fastq) file is like FASTA (.fasta) file + quality check data.

Click FASTQ link right side of SRR747784 that is the 1st Run data.

Now you can see the URL [5].

Download the 2 .fastq file.

$ cd YOUR_PROJECT_DIR

$ wget ftp://ftp.ddbj.nig.ac.jp/ddbj_database/dra/fastq/SRA067/SRA067162/SRX242800/SRR747784_1.fastq.bz2
$ wget ftp://ftp.ddbj.nig.ac.jp/ddbj_database/dra/fastq/SRA067/SRA067162/SRX242800/SRR747784_2.fastq.bz2

Extract the files.

$ ls -lh
total 239632
-rw-r--r--  1 jun.aruga  staff    57M Apr 22 09:44 SRR747784_1.fastq.bz2
-rw-r--r--  1 jun.aruga  staff    58M Apr 22 09:46 SRR747784_2.fastq.bz2

$ bunzip2 SRR747784_1.fastq.bz2
$ bunzip2 SRR747784_2.fastq.bz2

$ ls -lh
total 919552
-rw-r--r--  1 jun.aruga  staff   224M Apr 22 09:44 SRR747784_1.fastq
-rw-r--r--  1 jun.aruga  staff   224M Apr 22 09:46 SRR747784_2.fastq

Run fastqc

Download fastqc application from the web site [6]. As I am using Mac, I would download the Mac dmg file. Copy FastQC.app to /Applications to install it.

GUI

By click the installed, you can see the GUI application.

CUI command line

The command is installed in below path.

$ /Applications/FastQC.app/Contents/MacOS/fastqc --version
FastQC v0.11.7
$ export PATH=$PATH:/Applications/FastQC.app/Contents/MacOS

$ which fastqc
/Applications/FastQC.app/Contents/MacOS/fastqc

$ fastqc --version
FastQC v0.11.7
$ cd YOUR_PROJECT_DIR

$ mkdir qc_out

$ fastqc -o qc_out SRR747784_1.fastq SRR747784_2.fastq
Started analysis of SRR747784_1.fastq
Approx 5% complete for SRR747784_1.fastq
Approx 10% complete for SRR747784_1.fastq
...
Approx 95% complete for SRR747784_1.fastq
Analysis complete for SRR747784_1.fastq
Started analysis of SRR747784_2.fastq
Approx 5% complete for SRR747784_2.fastq
Approx 10% complete for SRR747784_2.fastq
...
Approx 95% complete for SRR747784_2.fastq
Analysis complete for SRR747784_2.fastq

The files are created in the qc_out/.

$ ls -l qc_out/
total 1848
-rw-r--r--@ 1 jun.aruga  staff  220242 Apr 22 10:22 SRR747784_1_fastqc.html
-rw-r--r--  1 jun.aruga  staff  228325 Apr 22 10:22 SRR747784_1_fastqc.zip
-rw-r--r--  1 jun.aruga  staff  221286 Apr 22 10:22 SRR747784_2_fastqc.html
-rw-r--r--  1 jun.aruga  staff  230648 Apr 22 10:22 SRR747784_2_fastqc.zip

See the .html file to see the result report.

f:id:happybirthday:20180422192059p:plain

Understanding the result report

The example reports "Good Illumina Data" and "Bad Illumina Data" in the FastQC official page [6] are helpful to understand the report.

There is also a tutorial video by FastQC project. www.youtube.com