The potential of hybridization capture to permit species identification in challenging leathers

Leather items, such as boots and wallets, are often made legally from mammal or reptile skins, but can also be made illegally from endangered species (sea turtles, Chinese alligator). In forensic wildlife casework, identifying the source species of leather evidence items can be particularly challenging, especially when items are small or when morphological characteristics only permit identification to a higher taxonomic level. When morphological species identifications cannot be made, wildlife forensic scientists often have to rely on genetic analysis to determine whether the item was made from a protected species. However, the series of chemicals used on animal skins in the leather-making process degrade DNA, making it highly fragmented and yielding low success (~30%) using standard PCR-based genetic methods. One potential solution to glean informative genetic information from severely compromised biological samples is to use new highly sensitive molecular methods, such as hybridization capture coupled with next-generation sequencing.

In this proposed study, a custom hybridization panel will be designed from full mitochondrial genomes to encompass ~50 species typically found in leather, especially those protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and the Endangered Species Act (ESA). DNA extracts from adjudicated leather material (n, 200) will be processed using an existing workflow, which briefly involves a) converting DNA into sequencing libraries, b) subjecting libraries to hybridization capture using the custom panel to enrich the mitochondrial genome from highly fragmented leather DNA, c) sequencing enriched mitochondrial DNA fragments using Illumina technology, and d) analyzing the resulting reads using an established data analysis pipeline. Success will be compared to results obtained from standard PCR-based methods, and a cost benefit analysis will be performed. 

While the proposed project aims to tackle challenging leather materials, the method could easily be incorporated into other casework evidence, such as caviar or cooked bushmeat, using an appropriate capture panel for the targeted species. If successful, the proposed project will initially benefit the partner laboratory (US Fish and Wildlife Service, Clark R Bavin National Fish and Wildlife Forensic Laboratory), which already has the expertise and equipment to implement the methods on casework samples. More broadly, there are several other wildlife forensic laboratories both domestically and internationally that are poised to transition emerging technologies that allow greater sensitivity, more success with degraded DNA, and better resolution of mixed samples, into forensic wildlife casework. CA/NCF