Trim low-quality bases and filter sequencing reads, short sequences produced by sequencing, using Trimmomatic, fastp, or cutadapt. Set quality and length thresholds, limit unknown bases, and process either single reads or paired reads for downstream analysis.
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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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查看安装方法安装前可以查看交给 AI 代理的指令内容。
Reference examples tested with: Trimmomatic 0.39+, cutadapt 4.4+, fastp 0.23+
Before using code patterns, verify installed versions match. If versions differ:
<tool> --version then <tool> --help to confirm flagsIf code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.
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.
trimmomatic PE with SLIDINGWINDOW and MINLEN optionsfastp --qualified_quality_phred 20 --length_required 50trimmomatic SE -phred33 \
input.fastq.gz output.fastq.gz \
LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36
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
| 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 |
# 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:<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
# 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
# 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
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
# 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
# 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
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 \
-a AGATCGGAAGAGC -A AGATCGGAAGAGC \
-q 20 -m 36 \
-o R1_trimmed.fq.gz -p R2_trimmed.fq.gz \
R1.fq.gz R2.fq.gz
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
| 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 |
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