FASTQ Quality Control and Trimming Guide

Introduction

The raw FASTQ reads sometimes contain adapter sequences and low-quality bases at the ends. These need to be removed before downstream analysis.

Step 1: Install Conda

Follow the instructions to install Miniconda on a Linux system:

mkdir -p ~/miniconda3
wget https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh -O ~/miniconda3/miniconda.sh
bash ~/miniconda3/miniconda.sh -b -u -p ~/miniconda3
rm ~/miniconda3/miniconda.sh

Refresh the terminal:

source ~/miniconda3/bin/activate

Initialize conda in all available shells:

~/miniconda3/bin/conda init --all

Step 2: Install and Use FastQC

Install FastQC:

conda create -n fastqc -c bioconda fastqc

Activate the environment:

conda activate fastqc

Run FastQC on all FASTQ files:

fastqc sub_samples2/*.fastq.gz -o .

Organize output files:

mkdir zip_files html_files
mv *.zip zip_files/
mv *.html html_files/

To visualize the HTML files:

exit
scp -r name@IP_address:/path/to/html_files ~/

Deactivate FastQC environment:

conda deactivate

Step 3: Trim Reads with Trim Galore

Install Trim Galore:

conda create -n trim-galore -c bioconda trim-galore

Activate the environment:

conda activate trim-galore

Trim paired-end reads with Illumina adapters:

trim_galore --paired --illumina \
sub_samples2/SRR10764405_1_subset.fastq.gz \
sub_samples2/SRR10764405_2_subset.fastq.gz

View trimming report:

less SRR10764405_1_subset.fastq.gz_trimming_report.txt

Bulk Trimming Options

Option 1: List file names manually

for file in \
sub_samples2/SRR10764405_1_subset.fastq.gz \
sub_samples2/SRR10764407_1_subset.fastq.gz \
sub_samples2/SRR10764408_1_subset.fastq.gz
do
    paired_file=${file/_1_subset.fastq.gz/_2_subset.fastq.gz}
    trim_galore --paired "$file" "$paired_file"
done

Option 2: Use wildcard to automatically detect file pairs

for file in sub_samples2/*_1_subset.fastq.gz
do
    trim_galore --paired "$file" "${file/_1_subset.fastq.gz/_2_subset.fastq.gz}"
done

Output Files:

• *_subset.fastq.gz_trimming_report.txt - Summary report.
• *_subset_val_1.fastq.gz - Trimmed read 1.
• *_subset_val_2.fastq.gz - Trimmed read 2.

Deactivate the Trim Galore environment:

conda deactivate