How to perform adapter trimming and quality control on paired-end Illumina reads with Trimmomatic

**Basic Trimmomatic PE command:** ```bash trimmomatic PE -threads 8 -phred33 sample_R1.fastq.gz sample_R2.fastq.gz sample_R1_paired.fastq.gz sample_R1_unpaired.fastq.gz sample_R2_paired.fastq.gz sample_R2_unpaired.fastq.gz ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:2:keepBothReads LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36 ``` **Parameters explained:** - `ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10`: adapter file, seed mismatch tolerance, palindrome score, simple clip score - `LEADING:3` / `TRAILING:3`: trim bases with quality <3 from ends - `SLIDINGWINDOW:4:15`: trim when 4-base window average quality drops below 15 - `MINLEN:36`: discard reads shorter than 36 bp after trimming **Adapter files** are bundled with Trimmomatic at `$CONDA_PREFIX/share/trimmomatic/adapters/`: - `TruSeq3-PE-2.fa` — Illumina TruSeq stranded kits (most common) - `NexteraPE-PE.fa` — Nextera XT, Nextera Flex **Quality check before and after:** ```bash # Before fastqc sample_R1.fastq.gz sample_R2.fastq.gz -o fastqc_before/ # After fastqc sample_R1_paired.fastq.gz sample_R2_paired.fastq.gz -o fastqc_after/ # Aggregate all samples multiqc fastqc_before/ fastqc_after/ -o multiqc_report/ ``` **Modern alternative: fastp** ```bash fastp -i R1.fastq.gz -I R2.fastq.gz -o R1_clean.fastq.gz -O R2_clean.fastq.gz --detect_adapter_for_pe -j report.json -h report.html -w 8 ``` fastp auto-detects adapters and is 3× faster than Trimmomatic.