ALLELE CHARACTERIZATION OF FIFTEEN SHORT TANDEM REPEAT LOCI OF NORTH AMERICAN GOLDEN (AQUILA CHYSAETOS) AND BALD (HALIAEETUS LEUCOCEPHALUS) EAGLES USING NEXT-GENERATION SEQUENCING

Purpose (a): The purpose of this study was to characterize by deep sequencing fifteen short tandem repeat (STR) loci in North American bald and golden eagles currently utilized by the US Fish and Wildlife Service. These STR loci are used for eagle identification in North America, however they were all developed from related species of European eagles. We hypothesized that using next-generation sequencing techniques we would be able to not only show concordance between allele calls using capillary electrophoresis (CE) methods and our new sequencing method, but also have an increased discrimination power of individuals by using this technique. Methods (b): Five samples of both bald and golden eagles were provided by the US Fish and Wildlife Service and deoxyribonucleic acid (DNA) was extracted from each using organic extraction. The current method of analysis through polymerase chain reaction (PCR) using fluorescent CE size based detection was adapted to create a protocol with increased specificity for the 15 sets of primers. Once the amplicon pool was developed, the amplicons were combined into a library using Library Prep Set for Ion Torrent™ kit for sequencing on the Ion Torrent™ Personal Genome Machine (PGM™) Sequencer® (Life Technologies™, Carlsbad, CA) with individual samples identified through barcodes embedded in the sequencing adaptors of the kit. After sequencing, the alleles were analyzed to identify the allele classification and determine the repeat structure of the STR motifs. The sequence data was used to determine if the sequences of North American population differed from existing reference sequences in GenBank®. Data was evaluated with the NextGENe® software using a virtual allelic ladder developed for each locus and contigs were assembled by anchoring the reads with the primer sequence. Results (c): Variants of alleles and allele distributions were recorded. The data revealed that the presence of sequence motif variation can increase the power of discrimination when using sequence analysis as compared to CE. Concordance was observed between the allele calls made using CE and with the sequence determined alleles. Conclusions (d): The method developed can be used to identify individual eagles with a higher discrimination power, which will be helpful in determining identity of individuals when needed for law enforcement investigations of harmed protected species such as bald and golden eagles.