Module 10: Structural Variant Calling: cuteSV, Sniffles2 & PBSV Algorithmic Tuning

1. The Taxonomy of Genomic Structural Variation

Structural variants (SVs) are genomic alterations involving at least $50\text{ base pairs}$ of DNA sequence. While single-nucleotide variants (SNVs) are more numerous, SVs account for over $75\%$ of all varying base pairs in any individual human genome.

The six fundamental SV classes include:

• Deletions (DEL): Loss of a contiguous chromosomal segment ($50\text{ bp} - 10\text{ Mb}$).
• Insertions (INS): Presence of a novel non-reference sequence or transposable element insertion (e.g. LINE-1, Alu).
• Duplications (DUP): Copy number gain, either tandemly duplicated or interspersed.
• Inversions (INV): Reversal of a chromosomal segment without net gain or loss of sequence.
• Translocations (BND): Fusion of segments between non-homologous chromosomes.
• Short Tandem Repeats (STR): Pathogenic repeat expansions of microsatellite loci.

2. Executing cuteSV on Long-Read Alignments

cuteSV uses a multi-tier clustering algorithm that analyzes soft-clipped read segments, CIGAR insertions/deletions, and supplementary alignments to achieve state-of-the-art precision and recall on ONT data:

# Call and genotype structural variants with cuteSV
mkdir -p ./sv_output/
cuteSV \
    aligned_sorted.bam \
    /references/GRCh38.fa \
    ./sv_output/hg002_cutesv.vcf \
    ./sv_output/work_dir/ \
    --threads 16 \
    --sample HG002_SAMPLE \
    --min_support 5 \
    --min_size 50 \
    --max_size 1000000 \
    --genotype \
    --retain_work_dir false

# Compress and index the output VCF
bgzip -c ./sv_output/hg002_cutesv.vcf > ./sv_output/hg002_cutesv.vcf.gz
tabix -p vcf ./sv_output/hg002_cutesv.vcf.gz

3. Sniffles2: Modern Population & Tandem Repeat SV Discovery

Sniffles2 provides specialized algorithms for mosaic SV detection, non-germline tumor SVs, and multi-sample cohort merging:

# Call SVs with Sniffles2 including tandem repeat profiling
sniffles \
    --input aligned_sorted.bam \
    --vcf ./sv_output/hg002_sniffles2.vcf.gz \
    --reference /references/GRCh38.fa \
    --threads 16 \
    --minsupport 5 \
    --minsvlen 50 \
    --tandem-repeats /annotations/human_GRCh38_no_alt_analysis_set.trf.bed