Improving accessibility of species identification for wildlife crime prosecution: Expanding WildSEQ for calcified and keratinised materials.

The growing need for simplified and accessible species identification methods is critical for supporting the prosecution of illegal wildlife trafficking. This requires the development of approaches suitable for implementation across both well-resourced and resource-limited jurisdictions. Here, we expand a previously developed targeted quantitative PCR (qPCR) and nanopore sequencing workflow (WildSEQ) to additional taxa including rhinoceros, hippopotamus, walrus, and water buffalo. Taxon-specific qPCR assays were developed to screen samples for multiple suspected species and this was combined with confirmatory sequencing for species identification. Assay performance was evaluated using both synthetic DNA and DNA extracted from casework-type samples, demonstrating high analytical sensitivity, with most assays detecting as little as 1 copy of target DNA. Across all samples, 90% were successfully detected by both qPCR and sequencing. Importantly, sequencing increased overall detection success rates to 95% by enabling species identification in samples that failed to meet qPCR calling criteria. Reliable species identification was achieved within 30 min of sequencing, demonstrating that sequencing time can be significantly reduced without compromising accuracy. The combined qPCR and sequencing approach enabled consistent species-level identification across taxa. Sequencing resolved cases of cross-reactivity and supported interpretation where qPCR amplification patterns alone were insufficient. The expanded WildSEQ workflow offers a rapid and accessible approach for species identification, with potential to support more widespread and routine implementation in wildlife forensic investigations. While further validation is required prior to operational use, this method has strong potential to facilitate increased testing capacity and strengthen enforcement efforts associated with illegal wildlife trade, including in resource-limited settings.