Whole Mitochondrial DNA Sequencing Using Fecal Samples from Domestic Dogs

Medical care for domestic dogs is now respected worldwide as being at a similar level to that of humans. We previously established a test method to determine whole mitochondrial DNA (mtDNA) using oral mucosal DNA that may be useful for medical care and welfare. However, the sample types tested in dogs are not limited to those obtained from the oral mucosa. Therefore, in the present study, we attempted to establish a test method to determine whole mtDNA sequences using feces, which represents the least invasive specimen. Two Japanese domestic dogs were used in the present study. DNA was extracted from approximately 100 mg of fresh feces from each dog, and PCRs were performed using four primer pairs that can amplify whole mtDNA. Following PCR, amplicons were pooled to create a DNA library using an experimental robot with an original program. Data were then acquired via NGS and data analysis was performed. The results showed that the whole mtDNA sequence of the two dogs was determined with high accuracy. Our results suggest that feces can be adapted for mitochondrial disease and individual identification testing and could serve as a useful testing method for the future medical care and welfare of domestic dogs.