Abstract:
Species identification is one of four key questions addressed in Wildlife Forensics, with DNA sequencing or barcoding one approach that could be applied. New technologies offer opportunities to overcome the limitations of existing species identification methods, which made them impractical for mainstream forensic laboratories. This thesis established a workflow combining Sanger and massively parallel sequencing techniques for forensic applications involving “difficult” non-human samples. Samples of processed meat, timber and wool carpet, reflecting a range of sample types, were used to test and develop a workflow. Specialised extraction methods, reported as optimal for the different sample types, were used. The mitochondrial cyt b and 16Smam, plastid trnL and trnL-trnF, and nuclear ITS2 universal markers were amplified in samples. Sanger sequencing of PCR products and subsequent species identification, the latter through BLAST algorithm query sequence alignment to a reference database, demonstrated the strengths and limitations of the overall workflow. Additional testing demonstrated the overall specificity, sensitivity and reproducibility of the established workflow. This workflow was applied to mixed samples of known and unknown composition (processed meats and Traditional East Asian Medicines) in combination with massively parallel sequencing. Using a novel USEARCH-Mothur/BLAST hybrid bioinformatic approach for species identification, the sensitivity and utility of this workflow in mixed sample authentication without a priori knowledge of composition was demonstrated. Encountered variation in ultimate identification success was attributed to the difficulty of samples in terms of DNA quality and quantity, biases of workflow stages, and the effects of species composition and manufacture. Although this study focussed primarily on New Zealand-relevant product authentication, this successful workflow combining sequencing techniques could be translated to applications in and provide insights for forensic casework where non-human samples present as potential evidential material.