Adapter Trimming

Remove sequencing adapters from reads using Cutadapt (precise, flexible) or Trimmomatic (paired-end optimized).

Common Adapter Sequences

Platform/Kit	Adapter	Sequence
Illumina TruSeq	Read 1 3'	AGATCGGAAGAGCACACGTCTGAACTCCAGTCA
Illumina TruSeq	Read 2 3'	AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
Nextera	Transposase	CTGTCTCTTATACACATCT
Small RNA	3' adapter	TGGAATTCTCGGGTGCCAAGG
Poly-A	Poly-A tail	AAAAAAAAAAAAAAAA

Cutadapt

Single-End Reads

# 3' adapter (most common)
cutadapt -a AGATCGGAAGAGC -o trimmed.fastq.gz sample.fastq.gz

# 5' adapter
cutadapt -g ACGTACGT -o trimmed.fastq.gz sample.fastq.gz

# Both ends
cutadapt -a ADAPTER1 -g ADAPTER2 -o trimmed.fastq.gz sample.fastq.gz

# Multiple adapters (tries each)
cutadapt -a ADAPTER1 -a ADAPTER2 -a ADAPTER3 -o trimmed.fastq.gz sample.fastq.gz

Paired-End Reads

# Basic paired-end
cutadapt -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCA \
         -A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT \
         -o trimmed_R1.fastq.gz -p trimmed_R2.fastq.gz \
         sample_R1.fastq.gz sample_R2.fastq.gz

# Short form for Illumina TruSeq (auto-detect)
cutadapt -a AGATCGGAAGAGC -A AGATCGGAAGAGC \
         -o trimmed_R1.fastq.gz -p trimmed_R2.fastq.gz \
         sample_R1.fastq.gz sample_R2.fastq.gz

Adapter Options

# Error rate (default 0.1 = 10% mismatches allowed)
cutadapt -a ADAPTER -e 0.15 -o out.fq in.fq

# Minimum overlap (default 3)
cutadapt -a ADAPTER -O 5 -o out.fq in.fq

# No indels in adapter alignment
cutadapt -a ADAPTER --no-indels -o out.fq in.fq

# Trim Ns from ends
cutadapt --trim-n -o out.fq in.fq

# Anchored adapters (must be at end)
cutadapt -a ADAPTER$ -o out.fq in.fq

Linked Adapters

# 5' adapter followed by 3' adapter (same read)
cutadapt -a ADAPTER1...ADAPTER2 -o out.fq in.fq

# Anchored 5' linked to 3'
cutadapt -a ^ADAPTER1...ADAPTER2 -o out.fq in.fq

Filtering After Trimming

# Minimum length (discard shorter)
cutadapt -a ADAPTER -m 20 -o out.fq in.fq

# Maximum length
cutadapt -a ADAPTER -M 150 -o out.fq in.fq

# Maximum N content
cutadapt -a ADAPTER --max-n 0.1 -o out.fq in.fq

# Discard trimmed reads
cutadapt -a ADAPTER --discard-trimmed -o out.fq in.fq

# Discard untrimmed reads
cutadapt -a ADAPTER --discard-untrimmed -o out.fq in.fq

Paired-End Filtering

# Both reads must pass minimum length
cutadapt -a ADAPT1 -A ADAPT2 -m 20 \
         -o R1.fq -p R2.fq in_R1.fq in_R2.fq

# Output too-short reads separately
cutadapt -a ADAPT1 -A ADAPT2 -m 20 \
         --too-short-output short_R1.fq --too-short-paired-output short_R2.fq \
         -o R1.fq -p R2.fq in_R1.fq in_R2.fq

Action Options

# Mask adapter instead of trim (replace with N)
cutadapt -a ADAPTER --action=mask -o out.fq in.fq

# Retain adapter but lowercase
cutadapt -a ADAPTER --action=lowercase -o out.fq in.fq

# Just find adapters, don't modify
cutadapt -a ADAPTER --action=none -o out.fq in.fq

Trimmomatic

Single-End Mode

trimmomatic SE -phred33 \
    input.fastq.gz output.fastq.gz \
    ILLUMINACLIP:adapters.fa:2:30:10

Paired-End Mode

trimmomatic PE -phred33 -threads 4 \
    input_R1.fastq.gz input_R2.fastq.gz \
    output_R1_paired.fastq.gz output_R1_unpaired.fastq.gz \
    output_R2_paired.fastq.gz output_R2_unpaired.fastq.gz \
    ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10

ILLUMINACLIP Parameters

ILLUMINACLIP:<fastaWithAdapters>:<seed>:<palindrome>:<simple>

# Parameters:
# seed - max mismatches in 16bp seed (usually 2)
# palindrome - threshold for palindrome match (usually 30)
# simple - threshold for simple match (usually 10)

# Example with all options
ILLUMINACLIP:adapters.fa:2:30:10:2:keepBothReads

Built-in Adapter Files

Trimmomatic includes adapter files:

• TruSeq2-SE.fa

  - TruSeq v2 single-end

• TruSeq2-PE.fa

  - TruSeq v2 paired-end

• TruSeq3-SE.fa

  - TruSeq v3 single-end

• TruSeq3-PE.fa

  - TruSeq v3 paired-end

• TruSeq3-PE-2.fa

  - TruSeq v3 PE (palindrome mode)

• NexteraPE-PE.fa

  - Nextera paired-end

Find Trimmomatic Adapters

# Find adapter directory
TRIMMOMATIC_JAR=$(which trimmomatic | xargs dirname)/../share/trimmomatic-*/adapters/

# Or with conda
ls $CONDA_PREFIX/share/trimmomatic-*/adapters/

Performance

# Cutadapt with multiple cores
cutadapt -j 8 -a ADAPTER -o out.fq in.fq

# Trimmomatic threads
trimmomatic PE -threads 8 ...

Verify Trimming

# Check adapter removal with FastQC
fastqc trimmed.fastq.gz

# Count reads before/after
zcat input.fastq.gz | wc -l
zcat trimmed.fastq.gz | wc -l

Related Skills

• quality-reports - Check adapter content with FastQC
• quality-filtering - Quality trimming after adapter removal
• fastp-workflow - Combined adapter and quality trimming