Combatting illegal trade in animal products by rapid onsite DNA sequencing

The illegal wildlife trade is a major threat to the survival of numerous species. A key challenge in combatting this trade is the difficulty in recognising protected species, particularly once processed. Traditionally, DNA-based species identification requires sending a sample to a laboratory where it is processed using expensive and bulky pieces of equipment, and the results are returned some time thereafter (days to weeks). It is therefore ill-suited to places lacking laboratory infrastructure. However, advances in sequencing technologies means that this process can now be carried out on-site. This project aimed to develop cheap and simple methods for DNA extraction, amplification, and sequencing using Oxford Nanopore Technologies’ MinION device. Primers were designed for four mitochondrial minibarcodes (cytochrome oxidase c subunit I, cytochrome b, 16S and 12S ribosomal RNA) to be as ‘universal’ as possible. The developed methods were successfully tested across 83 known vertebrate species and a wide range of sample types (hairs, feathers, blood, etc.), as well as more complex sample types including mixtures of both known and unknown compositions. The methods were also tested during a blind test at Twycross Zoo, and then in a molecular biology laboratory at the National University of Mongolia, Mongolia. The minibarcode tetraplex used proved robust to the presence of numts (nuclear mitochondrial DNA insertions), incomplete reference sequence data, and the limited intra-specific variation previously captured within available reference data. Additionally, new reference sequence data were generated by taking advantage of nanopore’s long-read sequencing capability to obtain whole mitochondrial genome sequences for an Altai marmot, a Siberian marmot, and a Daurian hedgehog, as well as the whole genome of a snow leopard. In the latter case, the genome was assessed for numt integration given frequent DNA-based misidentifications due to the co- or preferential amplification of numt sequences instead of the mitochondrial target barcode.