Quality Filtering

Trim low-quality bases and filter reads using Trimmomatic sliding window or fastp quality filtering.

Trimmomatic Quality Operations

Single-End Mode

trimmomatic SE -phred33 \
    input.fastq.gz output.fastq.gz \
    LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

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 \
    LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36

Trimmomatic Operations

Operation	Syntax	Description
LEADING	LEADING:Q	Remove leading bases below quality Q
TRAILING	TRAILING:Q	Remove trailing bases below quality Q
SLIDINGWINDOW	SLIDINGWINDOW:W:Q	Cut when W-bp window average < Q
MINLEN	MINLEN:L	Discard reads shorter than L
CROP	CROP:L	Cut read to max length L
HEADCROP	HEADCROP:N	Remove first N bases
AVGQUAL	AVGQUAL:Q	Drop read if average quality < Q
MAXINFO	MAXINFO:L:S	Balance length and quality
TOPHRED33	TOPHRED33	Convert to Phred33 encoding
TOPHRED64	TOPHRED64	Convert to Phred64 encoding

Common Trimmomatic Recipes

# Standard quality trimming
trimmomatic SE input.fq output.fq \
    SLIDINGWINDOW:4:20 MINLEN:36

# Aggressive 3' trimming
trimmomatic SE input.fq output.fq \
    TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:36

# Trim both ends, strict filtering
trimmomatic SE input.fq output.fq \
    LEADING:10 TRAILING:10 SLIDINGWINDOW:4:25 MINLEN:50

# Keep fixed length (for some tools)
trimmomatic SE input.fq output.fq \
    CROP:100 MINLEN:100

# Remove first 10 bases (e.g., random primers)
trimmomatic SE input.fq output.fq \
    HEADCROP:10 MINLEN:36

SLIDINGWINDOW Details

SLIDINGWINDOW:<windowSize>:<requiredQuality>

# Scan from 5' to 3'
# Cut when average quality in window drops below threshold
# Common settings: 4:15, 4:20, 5:20

# Conservative (keep more, lower quality)
SLIDINGWINDOW:4:15

# Moderate
SLIDINGWINDOW:4:20

# Strict (keep less, higher quality)
SLIDINGWINDOW:4:25

fastp Quality Filtering

Basic Quality Filtering

# Quality filtering (default Q15)
fastp -i in.fq -o out.fq

# Custom quality threshold
fastp -i in.fq -o out.fq -q 20

# Sliding window from 5' end
fastp -i in.fq -o out.fq --cut_front --cut_front_window_size 4 --cut_front_mean_quality 20

# Sliding window from 3' end
fastp -i in.fq -o out.fq --cut_tail --cut_tail_window_size 4 --cut_tail_mean_quality 20

# Aggressive right-side trimming (recommended)
fastp -i in.fq -o out.fq --cut_right --cut_right_window_size 4 --cut_right_mean_quality 20

fastp Quality Options

# Global mean quality filter
fastp -i in.fq -o out.fq -q 20 -e 25
# -q: per-base quality threshold
# -e: average quality threshold for entire read

# Unqualified bases threshold
fastp -i in.fq -o out.fq --unqualified_percent_limit 40
# Discard if >40% bases below quality threshold

# N base filtering
fastp -i in.fq -o out.fq -n 5
# Discard reads with >5 N bases

Paired-End with fastp

fastp -i R1.fq -I R2.fq -o out_R1.fq -O out_R2.fq \
    --cut_right \
    --cut_right_window_size 4 \
    --cut_right_mean_quality 20 \
    -q 20 -l 36

Length Filtering

# Trimmomatic
trimmomatic SE input.fq output.fq MINLEN:50

# fastp
fastp -i in.fq -o out.fq -l 50          # min length
fastp -i in.fq -o out.fq --length_limit 150  # max length

Cutadapt Quality Trimming

# Trim 3' end below Q20
cutadapt -q 20 -o out.fq in.fq

# Trim both ends
cutadapt -q 20,20 -o out.fq in.fq

# With minimum length
cutadapt -q 20 -m 36 -o out.fq in.fq

# Paired-end
cutadapt -q 20 -m 36 -o R1.fq -p R2.fq in_R1.fq in_R2.fq

Combined Adapter + Quality Trimming

Trimmomatic Full Pipeline

trimmomatic PE -threads 4 -phred33 \
    R1.fq.gz R2.fq.gz \
    R1_paired.fq.gz R1_unpaired.fq.gz \
    R2_paired.fq.gz R2_unpaired.fq.gz \
    ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10:2:keepBothReads \
    LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:36

Cutadapt Full Pipeline

cutadapt \
    -a AGATCGGAAGAGC -A AGATCGGAAGAGC \
    -q 20 -m 36 \
    -o R1_trimmed.fq.gz -p R2_trimmed.fq.gz \
    R1.fq.gz R2.fq.gz

Poly-G Trimming (NovaSeq/NextSeq)

NextSeq and NovaSeq use two-color chemistry, causing poly-G artifacts at read ends.

# fastp auto-detects and trims poly-G
fastp -i in.fq -o out.fq --trim_poly_g

# Disable auto-detection
fastp -i in.fq -o out.fq --disable_trim_poly_g

# Trimmomatic (manual approach)
# Add poly-G to adapter file

Quality Thresholds

Phred	Error Rate	Use Case
Q10	10%	Very lenient
Q15	3%	fastp default
Q20	1%	Common threshold
Q25	0.3%	Strict
Q30	0.1%	Very strict

Related Skills

• adapter-trimming - Remove adapters before quality filtering
• quality-reports - Check quality before/after filtering
• fastp-workflow - All-in-one preprocessing