This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Whole Mitochondrial DNA Sequencing Using Fecal Samples from Domestic Dogs
by
, , , , and
Takehito Sugasawa
1,2,*, 
Kieu D. M. Nguyen
3, 
Norihiro Otani
4,
Kiyoshi Maehara
5,
Fuka Kamiya
4,
Atsushi Hirokawa
4,
Tohru Takemasa
6,
Koichi Watanabe
6,
Takeki Nishi
7,
Ken Sato
7,
Suzuka Shimmura
7,
Yoichiro Takahashi
7 and
Yasuharu Kanki
1 1
Laboratory of Clinical Examination and Sports Medicine, Department of Clinical Medicine, Institute of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan
2
Department of Sports Medicine Analysis, Open Facility Network Office, Organization for Open Facility Initiatives, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan
3
Human Biology Program, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan
4
College of Medicine, School of Medicine and Health Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8577, Japan
5
Doctoral Program in Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan
6
Institute of Health and Sport Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8574, Japan
7
Department of Legal Medicine, Institute of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan
*
Author to whom correspondence should be addressed.
Animals 2024, 14(19), 2872; https://doi.org/10.3390/ani14192872 (registering DOI)
Submission received: 23 August 2024
/
Revised: 23 September 2024
/
Accepted: 27 September 2024
/
Published: 5 October 2024
(This article belongs to the Section Companion Animals)
Simple Summary
Several DNA tests are available for individual identification and mitochondrial diseases. Among them, the sequencing of whole mitochondrial DNA (mtDNA) is a beneficial method. Although DNA analysis requires a sample from the individual dog (e.g., blood or tissue), fecal samples are considered non-invasive and the least stressful form of sampling. In this study, we attempted to establish a method for sequencing whole mtDNA using small fecal samples (100 mg) from two domestic dogs. We also attempted to establish a method that is as accessible as possible to veterinary researchers. As a result, we were able to determine the samples’ whole mtDNA sequences with high accuracy. The method developed in this study shows that mtDNA from feces can be decoded and that this form of sampling provides a suitable sample for mtDNA decoding. The presented findings will inform future developments in veterinary medicine and animal welfare.
Abstract
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.
