Establishing a Sequencing Method for the Whole Mitochondrial DNA of Domestic Dogs

SIMPLE SUMMARY: The present study aims to establish a method of whole mitochondrial DNA (mtDNA) sequencing for domestic dogs. Prior to proceeding with the experiment, the collection of relevant DNA samples was essential. Therefore, we decided to use oral mucosa DNA, which can be collected non-invasively. A polymerase chain reaction (PCR) was performed using the DNA collected from six dogs raised in Japan and four primer pairs as specific to the mtDNA, after which the amplified products were sequenced using next-generation sequencing. As a result, the whole mtDNA obtained from all dogs was correctly sequenced. Thus, we determined that the method we used in our study may be useful for future research on dog-related medical care and welfare. ABSTRACT: In human beings, whole mitochondrial DNA (mtDNA) sequencing has been widely used in many research fields, including medicine, forensics, and genetics. With respect to the domestic dog (Canis lupus familiaris), which is commonly recognized as being an additional member of the traditional human family structure, research studies on mtDNA should be developed to expand and improve our collective knowledge of dog medicine and welfare as it seems that there is still room for further development in these areas. Moreover, a simple and robust method for sequencing whole mtDNA that can be applied to various dog breeds has not yet been described in the literature. In the present study, we aim to establish such a method for the whole mtDNA sequencing of the domestic dog. In the experiments we conducted, oral mucosa DNA samples obtained from six Japanese domestic dogs were used as a template. We designed four primer pairs that could amplify approximately 5 kbp from each region of the mtDNA and validated several PCR conditions. Subsequently, the PCR amplicons were pooled and subjected to library preparation. The sequencing of the libraries was performed using next-generation sequencing (NGS), followed by bioinformatics analysis. Our results demonstrate that the proposed method can be used to perform highly accurate resequencing. We believe that this method may be useful for future research conducted to better understand dog medicine and welfare.