Comprehensive Trimmomatic Usage Guide

---

Introduction to Trimmomatic

Trimmomatic is a versatile, fast, and multithreaded command-line tool designed for processing Illumina FASTQ data. It performs essential trimming tasks such as removing adapters, trimming low-quality bases, and filtering reads based on quality and length criteria. The software supports both single-end and paired-end data.

It provides robust methods for:

• Removing adapter sequences
• Trimming low-quality bases
• Filtering reads based on quality and length
• Supporting both single-end and paired-end sequencing data

This guide provides an in-depth tutorial on using Trimmomatic, detailing its features, usage modes, processing steps, and examples.

Installation and Prerequisites

System Requirements

• Java Runtime Environment (JRE)
• Sufficient computational resources
• Compressed input file support (gzip, bzip2)

Installation

Trimmomatic is distributed as a .jar file requiring Java. To install:

1. Ensure Java Runtime Environment (JRE) is installed.
2. Download the latest version of Trimmomatic from the official repository.
3. Save the trimmomatic-.jar file to your desired directory.

You can run Trimmomatic using the java -jar command.

Basic Command Structure

Trimmomatic supports two primary modes of operation:

Single-End Mode

java -jar trimmomatic.jar SE [options] \
<input_file> <output_file> \
<processing_steps>

Paired-End Mode

java -jar trimmomatic.jar PE [options] \
<input_file_1> <input_file_2> \
<output_paired_1> <output_unpaired_1> \
<output_paired_2> <output_unpaired_2> \
<processing_steps>

Application example PE mode

Recommended Command

java -jar trimmomatic-0.39.jar PE \
    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:2:30:10:2:True \
    LEADING:3 TRAILING:3 MINLEN:36

Alternative Command (More Sensitive)

java -jar trimmomatic-0.35.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

Processing Steps Breakdown

1. Remove adapters using TruSeq3-PE.fa
2. Remove leading low-quality bases (quality < 3)
3. Remove trailing low-quality bases (quality < 3)
4. Optional: Sliding window trimming (4-base window, quality threshold 15)
5. Drop reads shorter than 36 bases

Application example SE mode

java -jar trimmomatic-0.35.jar SE -phred33 \
    input.fq.gz output.fq.gz \
    ILLUMINACLIP:TruSeq3-SE:2:30:10 \
    LEADING:3 TRAILING:3 \
    SLIDINGWINDOW:4:15 MINLEN:36

Processing Steps

Trimmomatic offers multiple processing steps that can be applied in sequence:

ILLUMINACLIP: Adapter Removal

ILLUMINACLIP:<adapter_file>:<seed_mismatches>:<palindrome_clip_threshold>:<simple_clip_threshold> \
#Remove adapters (ILLUMINACLIP:TruSeq3-PE.fa:2:30:10)
ILLUMINACLIP:TruSeq3-PE.fa:2:30:10:2:True
#less sensitive for adapters
ILLUMINACLIP:TruSeq3-PE.fa:2:30:10

• Removes Illumina adapters
• Supports both simple and palindrome clipping strategies
• Recommended for most Illumina sequencing data

SLIDINGWINDOW: Quality-Based Trimming

SLIDINGWINDOW:<window_size>:<required_quality>
#Example
SLIDINGWINDOW:4:15
#Scan the read with a 4-base wide sliding window, cutting when the average quality per base drops below 15

• Scans reads with a sliding window
• Trims when average quality drops below threshold
• Prevents removal of entire reads due to single low-quality bases

LEADING: Trim Low-Quality Bases from Start

LEADING:<quality_threshold>
#Example
LEADING:3
Remove leading low quality quality 3

• Removes low-quality bases from the start of reads

TRAILING: Trim Low-Quality Bases from End

TRAILING:<quality_threshold>
#Example
TRAILING:3
Remove trailing low quality 3

• Removes low-quality bases from the end of reads

CROP: Limit Read Length

CROP:<length>

• Truncates reads to a specified maximum length

HEADCROP: Remove Fixed Number of Bases

HEADCROP:<length>

• Removes a specified number of bases from the start of reads

MINLEN: Minimum Read Length Filter

MINLEN:<length>
#Example
MINLEN:36

• Discards reads shorter than specified length
• Typically applied after other processing steps

Practical Example: Paired-End Processing

java -jar trimmomatic-0.32.jar PE \
    input_forward.fq.gz input_reverse.fq.gz \
    output_paired_forward.fq.gz output_unpaired_forward.fq.gz \
    output_paired_reverse.fq.gz output_unpaired_reverse.fq.gz \
    ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 \
    LEADING:3 \
    TRAILING:3 \
    SLIDINGWINDOW:4:15 \
    MINLEN:36

This example demonstrates:

• Adapter removal using TruSeq3 adapters
• Removing low-quality bases from start and end
• Sliding window quality trimming
• Minimum length filtering

Adapter Sequences

Choosing the Right Adapter File

• TruSeq2: For GAII machines
• TruSeq3: For HiSeq and MiSeq machines
• Verify adapter sequences using FASTQC’s “Overrepresented Sequences” report

Best Practices

• Always use the most recent adapter sequences
• Adjust parameters based on your specific sequencing platform
• Validate trimming results with quality control tools
• Consider downstream analysis requirements when setting thresholds

Performance Optimization

• Use -threads parameter for multi-core processing
• Supports automatic thread selection
• Quality encoding auto-detection since version 0.32

Troubleshooting

• Check input file formats (FASTQ, compressed)
• Verify adapter sequences
• Ensure Java version compatibility
• Review trimming logs for detailed processing information

Limitations and Considerations

• Adapter detection is a trade-off between sensitivity and specificity
• Short adapter fragments might remain undetected
• Performance varies with read length and quality

Always validate and optimize parameters for your specific dataset and research requirements.

---

References

Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics, btu170.

Download Trimmomatic manual