Agent skill

bio-read-qc-quality-filtering

Filter reads by quality scores, length, and N content using Trimmomatic and fastp. Apply sliding window trimming, remove low-quality bases from read ends, and discard reads below thresholds. Use when reads have poor quality tails or require minimum quality for downstream analysis.

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Install this agent skill to your Project

npx add-skill https://github.com/FreedomIntelligence/OpenClaw-Medical-Skills/tree/main/skills/bio-read-qc-quality-filtering

SKILL.md

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

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

Paired-End Mode

bash
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

bash
# 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

bash
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

bash
# 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

bash
# 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

bash
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

bash
# 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

bash
# 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

bash
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

bash
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.

bash
# 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

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