ALLELE CHARACTERIZATION OF TEN SHORT TANDEM REPEAT LOCI OF NORTH AMERICAN BEARS (URSIDS) USING NEXT-GENERATION SEQUENCING

Purpose (a): In the areas of conservation genetics and wildlife forensics, it is important to be able to accurately identify an individual and relate that individual back to the population from whence it came. With the low levels of genetic diversity possessed by some isolated North American bear populations, an identification method is needed that can provide higher resolution in short tandem repeat (STR) regions than current capillary electrophoresis assays. High resolution sequencing methods, such as those provided by next-generation sequencing (NGS) technologies, allow sequence motif changes within STR regions to be detected, whereas capillary electrophoresis only detects STR size. In this study, I hypothesized that when compared to capillary electrophoresis, NGS of STR loci would attain better genetic resolution among bear populations and thereby improve the accuracy of assigning an individual to its true population. Methods (b): Polymerase chain reaction (PCR) conditions were optimized for ten Ursid STR loci. An amplicon pool was generated and used to develop a library for NGS on the Ion Torrent™ Personal Genome Machine™ (PGM™) Sequencer (Life Technologies™, Carlsbad, CA). Deoxyribonucleic acid (DNA) barcode adaptors were ligated to the amplicons, thus allowing multiple samples to be sequenced in one run. Sequencing reads were aligned to a virtual ladder, first by sample via barcode, then by locus via primer. Sequence analysis was then performed using NextGENe® (SoftGenetics®, State College, PA) software. Results (c): Successful sequencing of ten loci for seven black bear (Ursus americanus) samples was carried out in one run. Allele call concordance was shown between capillary electrophoresis and NGS technologies. Variants within alleles (base changes) were evaluated and showed that NGS provided higher genetic resolution. Conclusions (d): In conclusion, NGS provided better genetic resolution than current capillary electrophoresis assays. Higher resolution has implications for increasing the accuracy of assigning an individual to its true population. Better assignment could improve genetic monitoring for conservation officers, as well as help wildlife forensic analysts improve evidentiary weight in wildlife crime cases that go to court.