Getting Started with Trimmomatic for Illumina Sequencing

High-throughput sequencing has become the cornerstone of modern genomics research. However, raw reads from Illumina platforms often contain technical artifacts—adapters, low-quality bases, or overly short reads—that can interfere with downstream analyses. That’s where Trimmomatic comes in.

Developed by Anthony M. Bolger, Marc Lohse, and Bjoern Usadel, Trimmomatic is a flexible and efficient tool specifically designed to preprocess Illumina NGS data. It is especially powerful when working with paired-end reads, ensuring data integrity throughout the trimming process.

✨ Key Features

• Adapter Removal
  Detects and removes adapter contamination to prevent false mapping and analysis errors.
• Quality Filtering
  Offers multiple quality trimming modes including:
  • Leading/Trailing trimming
  • Sliding window filtering
  • Maximum information-based trimming
• Minimum Length Enforcement
  Filters out short reads that could bias results.
• Cross-platform
  Requires Java (1.5 or higher), and works on Windows, macOS, and Linux.

Trimmomatic is open-source under the GPL v3 license and is maintained by the Usadel Lab.

⚙️ Installation

📦 Step 1: Install Java

Ensure Java is installed and available in your system’s path.

Ubuntu / Mint

sudo apt install default-jre

CentOS / Redhat

sudo yum install java-1.8.0-openjdk

Verify with:

java -version

🔧 Step 2: Install Trimmomatic

1. Download the latest .zip from the Trimmomatic website.
2. Extract the files into a directory.
3. Run using:

java -jar trimmomatic-0.39.jar [options]

🚀 Quick Start Command

Here’s a full example for paired-end data preprocessing:

java -jar trimmomatic-0.39.jar PE -phred33 \
input_forward.fq.gz input_reverse.fq.gz \
output_forward_paired.fq.gz output_forward_unpaired.fq.gz \
output_reverse_paired.fq.gz output_reverse_unpaired.fq.gz \
ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 \
LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

This command:

• Removes adapter sequences.
• Trims low-quality bases from the start and end.
• Performs sliding window trimming.
• Discards reads shorter than 36 bases.

🔧 Common Trimmomatic Commands

🧬 Adapter Removal

ILLUMINACLIP:adapter_sequences.fa:2:30:10

Removes known adapter sequences. adapter_sequences.fa contains the adapter list.

⚠️ Leading/Trailing Quality Trimming

LEADING:20 TRAILING:20

Trims bases below a quality threshold of 20 at both ends.

📉 Sliding Window Trimming

SLIDINGWINDOW:4:15

Scans the read and trims when the average quality in a 4-base window falls below 15.

🔪 Minimum Length Filter

MINLEN:36

Removes reads shorter than 36 bases after trimming.

✂️ Cropping

CROP:75

Truncates each read to the first 75 bases.

🧪 Why Use Trimmomatic?

• Improves mapping accuracy
• Removes artifacts that skew results
• Protects downstream tools from input errors
• Essential step before alignment, quantification, or variant calling

Its rich set of options makes Trimmomatic a go-to tool for preprocessing high-quality NGS data.

📚 Final Thoughts

Trimmomatic is more than a trimmer—it’s a reliable preprocessing toolkit that empowers researchers to clean and refine sequencing data efficiently. Its blend of flexibility, speed, and precision makes it an indispensable part of any Illumina NGS workflow.