Version Compatibility | Skills Pool

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Version Compatibility

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.

John-Wang-08090 스타2026. 3. 16.

직업
카테고리: 생물정보학

스킬 내용

Version Compatibility

Reference examples tested with: bcftools 1.19+, minimap2 2.26+, samtools 1.19+

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.

Long-Read SV Pipeline

"Detect structural variants from my long-read sequencing data" → Orchestrate minimap2 alignment, SV calling (Sniffles2/cuteSV), VCF merging across callers, annotation (AnnotSV), and visualization for ONT or PacBio data.

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

관련 스킬

# 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

# 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

samtools flagstat aligned.bam
samtools depth -a aligned.bam | awk '{sum+=$3} END {print "Average coverage:",sum/NR}'

# 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

# 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

# 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

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

# 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

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

#!/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)"