Agent skill

bio-workflows-longread-sv-pipeline

End-to-end workflow for detecting structural variants from long-read sequencing data. Covers ONT/PacBio alignment with minimap2 and SV calling with Sniffles or cuteSV. Use when detecting structural variants from long reads.

View SKILL.md on GitHub Repository
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Install this agent skill to your Project

npx add-skill https://github.com/majiayu000/claude-skill-registry/tree/main/skills/data/longread-sv-pipeline

SKILL.md

Long-Read SV Pipeline

Complete workflow for detecting structural variants from ONT or PacBio long-read data.

Workflow Overview

Long reads (ONT/PacBio)
    |
    v
[1. QC] ----------------> NanoPlot
    |
    v
[2. Alignment] ---------> minimap2
    |
    v
[3. SV Calling] --------> Sniffles / cuteSV
    |
    v
[4. Filtering] ---------> bcftools
    |
    v
[5. Annotation] --------> AnnotSV (optional)
    |
    v
Filtered SV VCF

Primary Path: minimap2 + Sniffles

Step 1: Quality Control

bash
# ONT reads QC
NanoPlot --fastq reads.fastq.gz \
    --outdir nanoplot_output \
    --threads 8

# Check key metrics
# - Read N50 should be >10kb
# - Mean quality >Q10
# - Total bases sufficient for coverage

Step 2: Alignment with minimap2

bash
# ONT reads
minimap2 -ax map-ont \
    -t 16 \
    --MD \
    -Y \
    reference.fa \
    reads.fastq.gz | \
    samtools sort -@ 4 -o aligned.bam

samtools index aligned.bam

# PacBio HiFi
minimap2 -ax map-hifi \
    -t 16 \
    --MD \
    -Y \
    reference.fa \
    reads.fastq.gz | \
    samtools sort -@ 4 -o aligned.bam

# PacBio CLR
minimap2 -ax map-pb \
    -t 16 \
    --MD \
    -Y \
    reference.fa \
    reads.fastq.gz | \
    samtools sort -@ 4 -o aligned.bam

QC Checkpoint: Check alignment stats

bash
samtools flagstat aligned.bam
samtools depth -a aligned.bam | awk '{sum+=$3} END {print "Average coverage:",sum/NR}'
  • Mapping rate >90%
  • Average coverage >10x for SV calling (>20x preferred)

Step 3: SV Calling with Sniffles

bash
# Sniffles2 (recommended)
sniffles \
    --input aligned.bam \
    --vcf svs.vcf.gz \
    --reference reference.fa \
    --threads 8 \
    --minsvlen 50

# With tandem repeat annotations (recommended)
sniffles \
    --input aligned.bam \
    --vcf svs.vcf.gz \
    --reference reference.fa \
    --tandem-repeats tandem_repeats.bed \
    --threads 8

Alternative: cuteSV

bash
# cuteSV (faster, good for ONT)
cuteSV \
    aligned.bam \
    reference.fa \
    svs.vcf \
    work_dir/ \
    --threads 8 \
    --min_size 50 \
    --genotype

bgzip svs.vcf
tabix svs.vcf.gz

Step 4: Filtering

bash
# Filter by quality and size
bcftools view -i 'QUAL>=20 && ABS(SVLEN)>=50' svs.vcf.gz -Oz -o svs.filtered.vcf.gz

# Filter by SV type
bcftools view -i 'SVTYPE="DEL" || SVTYPE="INS"' svs.filtered.vcf.gz -Oz -o del_ins.vcf.gz

# Filter by genotype
bcftools view -i 'GT="1/1" || GT="0/1"' svs.filtered.vcf.gz -Oz -o genotyped.vcf.gz

# Stats
bcftools stats svs.filtered.vcf.gz > sv_stats.txt

Step 5: Annotation (Optional)

bash
# AnnotSV for gene/clinical annotations
AnnotSV -SVinputFile svs.filtered.vcf.gz \
    -outputFile annotated_svs \
    -genomeBuild GRCh38

Multi-Sample SV Calling

bash
# Call SVs per sample
for sample in sample1 sample2 sample3; do
    sniffles --input ${sample}.bam \
        --snf ${sample}.snf \
        --reference reference.fa
done

# Merge and joint genotype
sniffles --input sample1.snf sample2.snf sample3.snf \
    --vcf merged_svs.vcf.gz \
    --reference reference.fa

Parameter Recommendations

Tool Parameter ONT PacBio HiFi
minimap2 -ax map-ont map-hifi
Sniffles --minsvlen 50 50
Sniffles --minsupport auto auto
cuteSV --min_size 50 50
cuteSV --min_support 3 3

SV Types Detected

Type Abbreviation Description
Deletion DEL Sequence removed
Insertion INS Sequence added
Duplication DUP Sequence copied
Inversion INV Sequence reversed
Translocation BND Breakend (interchromosomal)

Troubleshooting

Issue Likely Cause Solution
Few SVs Low coverage Increase sequencing depth
Many false positives Low quality reads Filter by QUAL, increase min support
Missing known SV Repeat region Use tandem repeat annotations
High breakend count Mapping artifacts Check alignment quality

Complete Pipeline Script

bash
#!/bin/bash
set -e

THREADS=16
READS="reads.fastq.gz"
REF="reference.fa"
SAMPLE="sample1"
OUTDIR="sv_results"

mkdir -p ${OUTDIR}/{qc,aligned,sv}

# Step 1: QC
echo "=== QC ==="
NanoPlot --fastq ${READS} --outdir ${OUTDIR}/qc -t ${THREADS}

# Step 2: Alignment
echo "=== Alignment ==="
minimap2 -ax map-ont -t ${THREADS} --MD -Y ${REF} ${READS} | \
    samtools sort -@ 4 -o ${OUTDIR}/aligned/${SAMPLE}.bam
samtools index ${OUTDIR}/aligned/${SAMPLE}.bam

echo "Alignment stats:"
samtools flagstat ${OUTDIR}/aligned/${SAMPLE}.bam

# Step 3: SV calling
echo "=== SV Calling ==="
sniffles --input ${OUTDIR}/aligned/${SAMPLE}.bam \
    --vcf ${OUTDIR}/sv/${SAMPLE}.vcf.gz \
    --reference ${REF} \
    --threads ${THREADS}

# Step 4: Filter
echo "=== Filtering ==="
bcftools view -i 'QUAL>=20' ${OUTDIR}/sv/${SAMPLE}.vcf.gz \
    -Oz -o ${OUTDIR}/sv/${SAMPLE}.filtered.vcf.gz
bcftools index ${OUTDIR}/sv/${SAMPLE}.filtered.vcf.gz

# Stats
bcftools stats ${OUTDIR}/sv/${SAMPLE}.filtered.vcf.gz > ${OUTDIR}/sv/stats.txt

echo "=== Complete ==="
echo "SVs: $(bcftools view -H ${OUTDIR}/sv/${SAMPLE}.filtered.vcf.gz | wc -l)"

Related Skills

  • long-read-sequencing/long-read-alignment - minimap2 details
  • long-read-sequencing/structural-variants - Sniffles, cuteSV options
  • long-read-sequencing/long-read-qc - NanoPlot metrics
  • variant-calling/structural-variant-calling - Short-read SV methods

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