In silico discovery of a nearly complete mitochondrial genome Numt in the dog (Canis lupus familiaris) nuclear genome.

Through bacterial cloning, a non-specific product co-amplified in a previous whole mitochondrial genome study of Canis lupus familiaris was identified as part of a Numt on chromosome 29 of the dog. Even though further analysis confirmed the fidelity of the mitochondrial genome sequencing results, it still highlighted the risk of Numt contamination. A computer-based search of the dog's nuclear genome for segments homologous to the mtDNA sequence revealed the extent of this risk. Over 150 Numts of various sizes were observed throughout all but two chromosomes, covering all positions of the mtDNA. One of the Numts on chromosome 11 even covered over 95 % of the entire dog mtDNA sequence. This comprehensive list of Numts was provided to assist researchers with the evaluation of dog mtDNA sequencing protocols for Numt co-amplification.

Length heteroplasmy of the polyC-polyT-polyC stretch in the dog mtDNA control region.

Previously, the mitochondrial control region of 214 Belgian dogs was sequenced. Analysis of this data indicated length heteroplasmy of the polyT stretch in the polyC-polyT-polyC stretch from positions 16661 to 16674. Nine polyC-polyT-polyC haplotype combinations were observed, consisting of seven major haplotypes (highest signal intensity) combined with minor haplotypes (lower signal intensity) one T shorter than the major haplotype in all but three dogs. The longer the polyT stretch, the smaller was the difference in signal intensity between the major and minor haplotype peaks. Additional sequencing, cloning, and PCR trap experiments were performed to further study the intra-individual variation of this mitochondrial DNA (mtDNA) region. Cloning experiments demonstrated that the proportion of clones displaying the minor haplotypes also increased with the length of the polyT stretch. Clone amplification showed that in vitro polymerase errors might contribute to the length heteroplasmy of polyT stretches with at least 10 Ts. Although major and minor polyC-polyT-polyC haplotypes did not differ intra-individually within and between tissues in this study, interpretation of polyT stretch variation should be handled with care in forensic casework.

Coding region SNP analysis to enhance dog mtDNA discrimination power in forensic casework.

The high population frequencies of three control region haplotypes contribute to the low discrimination power of the dog mtDNA control region. It also diminishes the evidential power of a match with one of these haplotypes in forensic casework. A mitochondrial genome study of 214 Belgian dogs suggested 26 polymorphic coding region sites that successfully resolved dogs with the three most frequent control region haplotypes. In this study, three SNP assays were developed to determine the identity of the 26 informative sites. The control region of 132 newly sampled dogs was sequenced and added to the study of 214 dogs. The assays were applied to 58 dogs of the haplotypes of interest, which confirmed their suitability for enhancing dog mtDNA discrimination power. In the Belgian population study of 346 dogs, the set of 26 sites divided the dogs into 25 clusters of mtGenome sequences with substantially lower population frequency estimates than their control region sequences. In case of a match with one of the three control region haplotypes, using these three SNP assays in conjunction with control region sequencing would augment the exclusion probability of dog mtDNA analysis from 92.9% to 97.0%.

Dog mitochondrial genome sequencing to enhance dog mtDNA discrimination power in forensic casework.

A Belgian dog population sample and several population studies worldwide have confirmed that only a limited number of mtDNA control region haplotypes is observed in the majority of dogs. The high population frequency of these haplotypes negatively impacts both the exclusion probability of dog mtDNA analysis and the evidential value of a match with one of these haplotypes in casework. Variation within the mtDNA coding region was explored to improve the discrimination power of dog mtDNA analysis. In the current study, the entire mitochondrial genome of 161 dogs was sequenced applying a quality assured strategy and resulted in a total of 119 different mitochondrial genome sequences. Our research was focused on those dogs with the six most common control region haplotypes from a previous Belgian population study. We identified 33 informative SNPs that successfully divide the six most common control region haplotypes into 32 clusters of mitochondrial genome sequences. Determining the identity of these 33 polymorphic sites in addition to control region sequencing in case of a match with one of these 6 control region haplotypes could augment the exclusion probability of forensic dog mtDNA analysis from 92.5% to 97.5%.

Reviewing population studies for forensic purposes: Dog mitochondrial DNA.

The identification of dog hair through mtDNA analysis has become increasingly important in the last 15 years, as it can provide associative evidence connecting victims and suspects. The evidential value of an mtDNA match between dog hair and its potential donor is determined by the random match probability of the haplotype. This probability is based on the haplotype's population frequency estimate. Consequently, implementing a population study representative of the population relevant to the forensic case is vital to the correct evaluation of the evidence. This paper reviews numerous published dog mtDNA studies and shows that many of these studies vary widely in sampling strategies and data quality. Therefore, several features influencing the representativeness of a population sample are discussed. Moreover, recommendations are provided on how to set up a dog mtDNA population study and how to decide whether or not to include published data. This review emphasizes the need for improved dog mtDNA population data for forensic purposes, including targeting the entire mitochondrial genome. In particular, the creation of a publicly available database of qualitative dog mtDNA population studies would improve the genetic analysis of dog traces in forensic casework.