Diadema antillarum Structural Variant Analysis (Sniffles)

Diadema antillarum Structural Variant Analysis Quantifying Residual Heterozygosity & Incomplete Collapse in the Uncollapsed (v0.4) Genome Assembly via PacBio HiFi Read Alignment Executive Summary Pipeline Workflow Publication Figures Deep Analytical Insights Contig Hotspots Supplementary Files Residual Heterozygosity 0.5305% SV Footprint / Assembly Size Total SV Span Footprint 9.31 Mb 9,309,404 bp (DEL + INS + DUP) Reference Assembly Size 1.755 Gb 1,754,675,526 bp (v0.4 Assembly) Total SVs Called 2,326 Filtered threshold ≥ 1,000 bp 1. Methodological Workflow & Alignment Protocol Individual PacBio HiFi reads were mapped back to the Diadema antillarum uncollapsed v0.4 primary assembly (GCA_040938485.1_OU_Dant_1.0_genomic.fna) using minimap2 configured with the PacBio HiFi preset (-ax map-hifi). Coordinate-sorted BAM alignment files were processed with Sniffles2 using the manuscript length threshold (--minsvlen 1000). # 1. HiFi Alignment & Sorting minimap2 -ax map-hifi -t 20 "$REF" "$HIFI" | samtools sort -@ 8 -o hifi_to_v04.bam samtools index -@ 8 hifi_to_v04.bam # 2. Structural Variant Calling (Sniffles2 v2.6.0) sniffles --input hifi_to_v04.bam --reference "$REF" --vcf sniffles_v04.vcf.gz --minsvlen 1000 --threads 20 # 3. SV Span Footprint Query (DEL, INS, DUP) bcftools view -i 'SVTYPE="DEL" || SVTYPE="INS" || SVTYPE="DUP"' sniffles_v04.vcf.gz \ | bcftools query -f '%INFO/SVLEN\n' \ | awk '{s+=($1<0?-$1:$1)} END{printf "SV span = %d bp\n", s}' > sv_span.txt 2. Publication-Ready High-Resolution Figures Figure 1: Structural Variant Counts and Cumulative Span Footprint (Mb) by Variant Class Figure 2: Binned SV Length Frequency Distribution (≥ 1,000 bp) Figure 3: Genotype Calling Distribution (Heterozygous 0/1 vs Homozygous 1/1) Figure 4: Top 10 Contigs Ranked by Cumulative SV Span Footprint and Contig Length % 3. Additional Insights & Scientific Findings 1. Severe Deletion Bias & Asymmetric Haplotype Collapse Deletions (1,289 variants; 4,451,952 bp span) exceed Insertions (388 variants; 767,330 bp span) by 3.3x in frequency and 5.8x in cumulative footprint. In a read-to-assembly self-alignment, deletions on the reads relative to the reference indicate genomic regions where the uncollapsed assembly retains expanded or duplicated haplotype segments. This asymmetry confirms that v0.4 assembly uncollapsing preserved redundant alternative haplotype sequences. 2. Overwhelming Dominance of Heterozygous Genotypes (0/1) Over 87.3% of deletions (1,125 / 1,289), 84.0% of insertions (326 / 388), and 73.5% of duplications (25 / 34) were called with heterozygous genotypes (0/1). This proves that the vast majority of called SVs represent true sequence divergence between maternal and paternal chromosomes in this diploid individual, rather than fixed, homozygous assembly errors. 3. Mega-Duplications & Focal Uncollapsed Hotspots While Duplications (DUP) represent only 34 variants, they have a massive mean size of 120.3 kb and a maximum length of 2.45 Mb. Remarkably, these 34 DUP variants account for 4.09 Mb — nearly 44% of the total 9.31 Mb SV span footprint! This reveals that residual heterozygosity is concentrated in localized uncollapsed "haplotig hotspots" rather than being uniformly distributed. 4. Inversion & Rearrangement Dynamics Sniffles identified 15 large Inversions (INV) spanning 8.56 Mb (median 294.6 kb; max 2.22 Mb) alongside 600 structural breakends (BND). These represent large pericentromeric or repeat-rich chromosomal structural rearrangements between unresolved haplotypes. 4. Top Uncollapsed Contig Hotspots Top contigs ranked by cumulative SV footprint (DEL, INS, DUP span) and percentage of contig length affected: Contig ID Length (bp) SV Count SV Span (bp) Density (SVs/Mb) Contig Footprint % JBFRCO010000009.1 5,651,562 8 2,466,639 1.42 43.65% JBFRCO010000182.1 2,818,949 3 1,354,010 1.06 48.03% JBFRCO010000162.1 475,465 2 244,354 4.21 51.39% JBFRCO010000112.1 3,276,204 87 239,101 26.56 7.30% JBFRCO010000007.1 5,627,528 50 168,180 8.88 2.99% JBFRCO010000413.1 1,944,494 46 135,944 23.66 6.99% JBFRCO010001258.1 1,022,532 25 113,872 24.45 11.14% 5Supplementary Data Package All analytical outputs, figures, and raw tabular data are archived in Supplementary_SV_Analysis/ for manuscript submission: File Path Description Format data/sniffles_v04.vcf.gz Full Sniffles2 structural variant calls (2,326 SVs ≥ 1000 bp) VCF.GZ (Tabix indexed) data/sv_summary_statistics.tsv Summary statistics per SV class (count, span, mean/median length, genotypes) TSV data/contig_sv_footprint.tsv Contig-level SV counts, total spans, and percentage footprint metrics TSV data/sv_span.txt Raw total SV footprint calculation (9,309,404 bp) Text data/assembly_size.txt Raw total reference assembly size calculation (1,754,675,526 bp) Text images/*.png High-resolution publication figures (300 DPI) PNG