bio-read-qc-quality-filtering

Version Compatibility

Reference examples tested with: Trimmomatic 0.39+, cutadapt 4.4+, fastp 0.23+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Quality Filtering

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

"Filter reads by quality" → Remove low-quality bases and discard reads below quality/length thresholds.

• CLI: trimmomatic PE with SLIDINGWINDOW and MINLEN options
• CLI: fastp --qualified_quality_phred 20 --length_required 50

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