How to Operate FastQC on Windows

1. Download FastQC

1. Download FastQC from the official website:
   https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
2. Install a suitable Java Runtime Environment (JRE). You can find a link to a suitable Java version on the FastQC website, or you can install any standard JRE (version 1.8 or above should work).

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2. Manual File Import and Execution

1. Extract the FastQC zip file to any convenient location on your computer.
   For example: C:\Tools\FastQC

2. Run FastQC via the GUI (using run_fastqc.bat):

   • In the extracted FastQC folder, look for a .bat file named run_fastqc.bat (or fastqc.bat in some distributions).
   • Double-click this .bat file to launch a simple graphical interface.
   • You can then manually select one or more FASTQ files to analyze.

3. Run FastQC via command line (using fastqc.exe):

   • Inside the extracted folder, you will see a file named fastqc.exe (or fastqc).
   • Open Command Prompt (or PowerShell), navigate to the FastQC directory, and enter a command such as:

     fastqc C:\path\to\your\data\sample1.fastq C:\path\to\your\data\sample2.fastq

     Note: On Windows, you might need to run fastqc.exe directly, or include the .exe extension if it’s not recognized automatically.

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3. Analyzing an Entire Folder of FASTQ Files with RUN_FastQC_Windows.R

Use the R script in this repository named RUN_FastQC_Windows.R that runs FastQC on all FASTQ files within a specified folder:

1. Open the script RUN_FastQC_Windows.R in any text editor (e.g., RStudio or Notepad).

2. Modify the following within the script:

   • FastQC executable path: Ensure the script points to the fastqc.exe file inside your extracted FastQC folder.

     fastqc_path <- "C:/Tools/FastQC/fastqc.exe"

   • Target folder path: Update the folder path containing your FASTQ files.

     data_folder <- "C:/Data/FASTQ_Files/"

3. Run the script in R or RStudio (or use Rscript from the command line):

   Rscript RUN_FastQC_Windows.R
   

This script will execute FastQC on every .fastq.gz file found in the designated folder.

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4. Summarizing Results with Sum_FastQC.R

After running FastQC on all your FASTQ files, you can use the script Sum_FastQC.R to automatically parse each _fastqc.zip result, extract the summary.txt files inside, and consolidate all QC metrics into a single table. The script then:

• Transforms the data into a wide-format spreadsheet (with metrics as rows and sample names as columns).
• Exports a standard CSV file (e.g., FastQC_summary.csv) for easy viewing.
• Generates a color-coded Excel file (e.g., FastQC_summary_colored.xlsx) where PASS, FAIL, and WARN cells are highlighted in different colors (green, red, and orange, respectively).

This consolidated summary allows you to quickly assess each QC metric’s status across all samples, without having to open individual FastQC reports.

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5. Adjusting Thresholds in limits.txt

If you need to change the default thresholds for QC indicators (e.g., minimum quality score, adapter contamination limits), you can modify the limits.txt file:

1. Locate the configuration folder in your FastQC installation. It may be at:

   C:\Tools\FastQC\fastqc_v0.12.1\FastQC\Configuration\limits.txt

(Adjust the path as needed, depending on where you extracted FastQC.)

2. Open limits.txt in a text editor.
3. Find the parameter(s) you need to change and edit their values (e.g., minimum per-base quality, maximum adapter contamination threshold).
4. Save the file, and re-run FastQC to apply the new thresholds.

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6. Additional Tips

• Check Java installation: If FastQC fails to launch, verify that Java is installed and that its path is correctly set in your system environment variables.
• GUI mode (optional): In some FastQC distributions for Windows, there is a .bat file (e.g., fastqc.bat). Double-clicking this can open a simple graphical interface, which allows you to select files manually.
• FastQC outputs: By default, FastQC creates a *_fastqc.html summary report and a *_fastqc.zip file for each input FASTQ. You can open the .html in any web browser to see the graphical summary.