Species identification is a key task in non-human forensic genetics, with applications ranging from wildlife investigations to alimentary fraud detection and food security. Currently, the gold standard for genetic species identification is Sanger sequencing, however, this technique shows some limitations- including high equipment requirements and the inability to analyse mixed samples - that could be addressed using NGS technologies such as nanopore sequencing.

This study presents an integrative nanopore-based workflow for taxonomic identification in forensic contexts. The wet lab stage involves a multiplex PCR which amplifies four mtDNA markers for species identification purposes (cytochrome c oxidase subunit I, cytochrome b, 12S rRNA, and 16S rRNA), designed to provide resolution across diverse taxa, followed by library preparation and sequencing on the portable MinION device. The bioinformatic stage combines metagenomic classification tools available within the EPI2ME workflows with the generation of a consensus sequence, enabling taxonomic assignment and compositional characterization of the analysed samples.

Samples representing forensic casework-like scenarios and food industry products, reflecting the variability of animal-derived materials, were evaluated using the workflow, which showed a clear advantage over the Sanger approach in the analysis of mixed samples. Furthermore, the accessibility of the technology, combined with the study design, ensures that the workflow can be applied to diverse forensic applications with only minor adjustments.

Authors

• Lucía Casanova-Adán (Forensic Genetics Unit, Institute of Forensic Sciences, Universidade de Santiago de Compostela, Spain)
• Kyle Ewart (Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Easter Bush Campus, EH25 9RG, United Kingdom)
• Javier González-Bao (Forensic Genetics Unit, Institute of Forensic Sciences, Universidade de Santiago de Compostela, Spain)
• Adrián Ambroa-Conde (Forensic Genetics Unit, Institute of Forensic Sciences, Universidade de Santiago de Compostela, Spain)
• Amaia Cabrejas-Olalla (Forensic Genetics Unit, Institute of Forensic Sciences, Universidade de Santiago de Compostela, Spain)
• Amelia Rodríguez-López (Forensic Genetics Unit, Institute of Forensic Sciences, Universidade de Santiago de Compostela, Spain)
• Ana Mosquera-Miguel (Forensic Genetics Unit, Institute of Forensic Sciences, Universidade de Santiago de Compostela, Spain)
• Rob Ogden (Royal (Dick) School of Veterinary Studies and the Roslin Institute, University of Edinburgh, Easter Bush Campus, United Kingdom)
• María Victoria Lareu (Forensic Genetics Unit, Institute of Forensic Sciences, Universidade de Santiago de Compostela, Spain)
• María de la Puente (Forensic Genetics Unit, Institute of Forensic Sciences, Universidade de Santiago de Compostela, Spain)