Developing Massively Parallel Sequencing multiplexes for Birds of Prey: A pilot study for non-human forensics and conservation biology

The persecution of birds of prey, including poaching, trapping, poisoning and shooting, is a persistent and highly damaging problem in the UK despite it being a national wildlife crime priority since 2009. Prosecution rates for these crimes are currently very low but DNA profiling can be used to address questions of parentage and identity which can serve as important evidence in wildlife crime cases. In human casework, DNA profiling by capillary electrophoresis (CE) of short tandem repeat (STR) loci is very well established but more recently, the adoption of massively parallel sequencing (MPS) technology is being increasingly applied to overcome some limitations of CE. This study is a pilot for developing MPS STR multiplexes for non-human forensics. Given their conservation value and the requirement for improved DNA testing for casework, raptors provide an ideal model. Novel STRs were identified from the genome sequences of the peregrine falcon, white-tailed eagle and golden eagle. As the Accipitridae family (hawks, kites and eagles) diverged from the Falconidae family (falcons) ~85 million years ago, two separate marker panels were developed. CE multiplexes were designed for both panels and screening across a wide range of species demonstrated that they are largely informative, independent and capture a wide range of interspecific and intraspecific variation. Multiplexes supplemented with some previously published markers were also developed for MPS which revealed additional alleles through variation in the repeat structure and flanking sequence. Results obtained via CE and MPS were concordant. The novel plexes were tested on real casework samples and gave concordant results to the original single locus minisatellite profiling markers used in the case. The multiplexes developed here also serve as a valuable tool for conservation genetics and population monitoring in raptors, as demonstrated by a novel study of the red kite.