High‐Fidelity Whole‐Genome Sequencing of White Spot Syndrome Virus From Infected Shrimp via Iodixanol Density Gradient Centrifugation and Enzymatic Host Depletion

ABSTRACT White Spot Disease (WSD), caused by White Spot Syndrome Virus (WSSV; Whispovirus xiabaidian ), inflicts catastrophic losses on global shrimp aquaculture. Characterizing strain‐level virulence variations requires comparative genomics; however, the lack of continuous crustacean cell lines severely constrains direct sequencing from infected tissues due to overwhelming host DNA background. Here, we established an efficient particle purification protocol coupling iodixanol density gradient ultracentrifugation with enzymatic nuclease digestion to selectively deplete host nuclear and mitochondrial DNA. The developed protocol enriched target WSSV reads to > 70% across both Illumina MiSeq and Oxford Nanopore MinION platforms while virtually eliminating coverage bias in non‐amplified libraries. Stepwise downsampling demonstrated that our reference‐guided assembly pipeline reliably reconstructs complete ~300‐kbp genomes from as little as 5–10 Mbp of sequencing data, enabling multiplexed sequencing at approximately $10 USD per isolate. Applying this optimized framework, we successfully resolved the complete genomes of two novel field isolates from Thailand (TH‐W11) and the Philippines (PHR3‐ZS‐M101724, representing the first complete WSSV genome from the country), uncovering distinct phylogenetic lineages and large genomic deletions (~5.6–5.8 kbp), which were experimentally confirmed by flanking PCR. This cost‐effective platform provides a standardized pipeline for high‐throughput genomic surveillance and molecular epidemiology of emerging WSSV isolates.