Reducing the Number of Mismatches between Hairs and Buccal References When Analysing mtDNA Heteroplasmic Variation by Massively Parallel Sequencing.

In forensics, mitochondrial DNA (mtDNA) analysis is foremost applied to rootless hairs often lacking detectable nuclear DNA. Sanger sequencing is the routine mtDNA method in most forensic laboratories, even though interpretation of mixed samples and heteroplasmic sites can be challenging. Individuals may hold cells with low-level heteroplasmy variants below the detection threshold and other cells where this minor variant is the major one. This difference may be interpreted as a mismatch between reference and evidentiary trace samples, such as buccal specimens and rootless hairs. Such mismatches may be solved by Massively Parallel Sequencing (MPS), allowing more sensitive quantitative analysis for mixed positions than Sanger. The mtDNA control region was analysed in buccal reference samples from 26 individuals and 475 corresponding hairs by MPS and compared to Sanger sequencing data generated on the same samples. With MPS, mixed contributions down to 3% were regarded, leading to a substantial increase in the frequency of heteroplasmy. Our results demonstrate that previously reported mismatches between buccal reference and hair shaft samples by Sanger are detected as low-level heteroplasmy by MPS. A detailed overview of buccal and hair heteroplasmy is provided and implications for MPS-based mtDNA analysis in the context of forensic cases are discussed.

Base specific variation rates at mtDNA positions 16093 and 16183 in human hairs.

Small variations between haplotypes detected in different tissues from the same individual have been previously described. These differences complicate the interpretation of mtDNA results in real forensic casework. mtDNA haplotypes detected in hair strands collected at the crime scene have to be frequently compared with haplotypes of reference samples (buccal swabs) from victims or suspects. Nucleotide position 16093 is a well-known hot spot where differences can accumulate between different tissues of the same individual. Intra individual variation was also detected at positions 16182 and 16183 in haplotypes showing an uninterrupted HV1 poly-C stretch (with 16189C). In order to better characterize the type of variation in these positions between buccal cells and hair strands from the same individual, we have performed Sanger sequencing in 25-28 hair strands (411 in total) from 15 individuals showing either an uninterrupted HV1 polyC-stretch (16189C) or 16093C/Y in their buccal cells. The results have been evaluated by also taking into account our previous results published in [19]. We have found that no variation among hair strands was detected in individuals showing T16093 in buccal cells, while variation in hair strands (T16093, 16093C and 16093Y) were detected in individuals showing 16093C or 16093Y in buccal cells. Regarding nucleotide positions 16182 and 16183 in combination with an uninterrupted polyC-stretch, no variation was detected in hairs from individuals showing A16182 16183C in their buccal cells. In contrast, individuals A16182 A16183 showed hair strands with A16182 16183 M and A16182 16183C. And finally, individuals with 16182C 16183C showed some variation in a small amount of their hair strands (some hairs with 16182 M 16183C). These results can be relevant for forensic practitioners when comparing reference samples with hair strands, which is the type of sample most tested by using mtDNA analysis in forensic casework.

Results of a collaborative study on DNA identification of aged bone samples.

AIM: A collaborative exercise with several institutes was organized by the Forensic DNA Service (FDNAS) and the Institute of the Legal Medicine, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic, with the aim to test performance of different laboratories carrying out DNA analysis of relatively old bone samples. METHODS: Eighteen laboratories participating in the collaborative exercise were asked to perform DNA typing of two samples of bone powder. Two bone samples provided by the National Museum and the Institute of Archaelogy in Prague, Czech Republic, came from archeological excavations and were estimated to be approximately 150 and 400 years old. The methods of genetic characterization including autosomal, gonosomal, and mitochondrial markers was selected solely at the discretion of the participating laboratory. RESULTS: Although the participating laboratories used different extraction and amplification strategies, concordant results were obtained from the relatively intact 150 years old bone sample. Typing was more problematic with the analysis of the 400 years old bone sample due to poorer quality. CONCLUSION: The laboratories performing identification DNA analysis of bone and teeth samples should regularly test their ability to correctly perform DNA-based identification on bone samples containing degraded DNA and potential inhibitors and demonstrate that risk of contamination is minimized.

Local populations and inaccuracies: Determining the relevant mitochondrial haplotype distributions for North West European cats.

Typing of different portions of the feline mitochondrial control region has illustrated pronounced differences in haplotype distributions between cats from the Netherlands and other parts of the world. To gain a better understanding of the haplotype distribution of North West Continental Europe, 605bp of mitochondrial DNA was typed from randomly selected cats from the Netherlands (N=146), Belgium (N=64) and South West Germany (N=128). The genetic differences between these randomly sampled European populations correlate to the geographical distances, with the Dutch and the South West German populations furthest apart and the Belgian population as an intermediate (Fst values 0.01-0.03). Comparison of North West European mainland distributions to published feline mitochondrial haplotype distributions illustrated moderate to large genetic differentiation (Fst values 0.01-0.32). In this comparison, the correlation between geographical and genetic distance was absent, leading to founder effects and human impact on cat population structure and dispersion being considered as important parameters. When an accurate estimation of feline haplotype distribution is required in forensics, care should be taken when deciding whether extrapolating the frequency data from a certain source to a larger area (country/continent) is justified or whether additional typing of local populations is necessary. This may differ from case to case as local frequencies can be relevant, but can also be deceitful. To improve the applicability of forensic feline mitochondrial DNA studies, documentation and publishing of sampling strategies is advised, as is the implementation of measures to help eliminate potentially erroneous haplotypes.

Organic extraction of bone lysates improves DNA purification with silica beads.

In our standard protocol for DNA extraction from skeletal remains of unidentified bodies, bone lysates resulting from decalcification and Proteinase K treatment were purified with the DNA IQ™ Casework Pro Kit for Maxwell®16 automate (Promega, WI). Despite its success in the majority of cases, the DNA purification with paramagnetic silica beads failed in some challenging samples. This failure in DNA recovery was often associated with filter clogging during the required volume reduction of the lysate to enable loading on the automate. Two modifications to the standard method were tested for a more efficient filtering and purification. Adding collagenase to the lysate reduced the filter lead time but did not enhance DNA yield, while organic extraction of the crude lysate solved the filter clogging and resulted in successful DNA purification. The modified method in which a phenol treated lysate was loaded on the automate resulted in successful STR-profiling of the skeletal remains of all 13 unidentified bodies tested, which showed a wide variety in post mortem interval and preservation conditions. The variation in DNA yield between the 28 samples tested showed the importance of bone type selection and multiple sampling in successful STR-profiling of skeletal remains. Despite the disadvantages inherent to phenol, the organic extraction of crude bone lysates enhanced the efficiency of DNA purification with paramagnetic silica beads. The combined method of organic extraction and purification with silica beads resulted in STR-profiling of challenging bone samples.

Hairy matters: MtDNA quantity and sequence variation along and among human head hairs.

Hairs from the same donor have been found to differ in mtDNA sequence within and among themselves and from other tissues, which impacts interpretation of results obtained in a forensic setting. However, little is known on the magnitude of this phenomenon and published data on systematic studies are scarce. We addressed this issue by generating mtDNA control region (CR) profiles of >450 hair fragments from 21 donors by Sanger-type sequencing (STS). To mirror forensic scenarios, we compared hair haplotypes from the same donors to each other, to the corresponding buccal swab reference haplotypes and analyzed several fragments of individual hairs. We also investigated the effects of hair color, donor sex and age, mtDNA haplogroup and chemical treatment on mtDNA quantity, amplification success and variation. We observed a wide range of individual CR sequence variation. The reference haplotype was the only or most common (≥75%) hair haplotype for most donors. However, in two individuals, the reference haplotype was only found in about a third of the investigated hairs, mainly due to differences at highly variable positions. Similarly, most hairs revealed the reference haplotype along their entire length, however, about a fifth of the hairs contained up to 71% of segments with deviant haplotypes, independent of the longitudinal position. Variation affected numerous positions, typically restricted to the individual hair and in most cases heteroplasmic, but also fixed (i.e. homoplasmic) substitutions were observed. While existing forensic mtDNA interpretation guidelines were found still sufficient for all comparisons to reference haplotypes, some comparisons between hairs from the same donor could yield false exclusions when those guidelines are strictly followed. This study pinpoints the special care required when interpreting mtDNA results from hair in forensic casework.

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.

Models and implementation for relationship problems with dropout.

Allelic dropout in relationship problems may commonly appear in areas such as disaster victim identification and the identification of missing persons. If dropout is not accounted for, the results may be incorrect interpretation of profiles, loss of valuable information and biased results. In this paper, we explore different models for dropout in kinship cases and present an efficient implementation for one of the models. The implementation allows for dropout to be handled simultaneously with phenomena like silent alleles and mutations that may also cause discordances in relationship data, in addition to subpopulation correction. The implemented dropout model is freely available in the new version of the Familias software. The concepts and methods are illustrated on real and simulated data.

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%.

Identification of forensically important Sarcophaga species (Diptera: Sarcophagidae) using the mitochondrial COI gene.

The identification of species of the forensically important genus Sarcophaga is very difficult and requires strong taxonomic expertise. In this study, we sequenced the mitochondrial cytochrome c oxidase subunit I (COI) gene of 126 specimens of 56 W European Sarcophaga species and added GenBank data to our database to yield a total dataset of 270 COI sequences from 99 Sarcophaga species to evaluate the COI gene as a molecular diagnostic tool for species identification in this genus. Using two simple criteria (Best Match, BM and Best Close Match, BCM), we showed that the identification success using a mini-barcode region of 127 bp was very low (80.7-82.5 %) and the use of this region is not recommended as a species identifier. In contrast, identification success was very high using the standard barcode region (658 bp) or using the entire COI region (1,535 bp) (98.2-99.3 %). Yet, there was a low interspecific sequence divergence (<2 %) in six species groups so that for 16 out of the 99 species (nine of which are of forensic importance), the use of COI barcodes as species identifier should be done with care. For these species, additional markers will be necessary to achieve a 100 % identification success. We further illustrate how such reference databases can improve local reference databases for forensic entomologists.

DNA barcoding and the differentiation between North American and West European Phormia regina (Diptera, Calliphoridae, Chrysomyinae).

Phormia regina (the black fly) is a common Holarctic blow fly species which serves as a primary indicator taxon to estimate minimal post mortem intervals. It is also a major research model in physiological and neurological studies on insect feeding. Previous studies have shown a sequence divergence of up to 4.3% in the mitochondrial COI gene between W European and N American P. regina populations. Here, we DNA barcoded P. regina specimens from six N American and 17 W European populations and confirmed a mean sequence divergence of ca. 4% between the populations of the two continents, while sequence divergence within each continent was a ten-fold lower. Comparable mean mtDNA sequence divergences were observed for COII (3.7%) and cyt b (5.3%), but mean divergence was lower for 16S (0.4-0.6%). Intercontinental divergence at nuclear DNA was very low (≤ 0.1% for both 28S and ITS2), and we did not detect any morphological differentiation between N American and W European specimens. Therefore, we consider the strong differentiation at COI, COII and cyt b as intraspecific mtDNA sequence divergence that should be taken into account when using P. regina in forensic casework or experimental research.

Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France.

Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available. We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems - BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes.

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.

Why is the molecular identification of the forensically important blowfly species Lucilia caesar and L. illustris (family Calliphoridae) so problematic?

Species of the fly genus Lucilia are commonly used in forensic investigations to estimate the postmortem interval (PMI). Two close-related species Lucilia caesar and L. illustris are difficult to identify. Previous studies showed that the mitochondrial cytochrome c oxidase subunit I (COI) marker could be used to identify many Lucilia species. However, mixed results were obtained for L. caesar and L. illustris due to some European specimens showing identical haplotypes. Here, we investigated 58 new European male specimens of L. illustris and L. caesar whose morphological identifications were checked and for which COI fragments were sequenced. In addition, two other mitochondrial (cytochrome c oxidase subunit II and 16S) and two nuclear (internal transcribed spacer 2 and 28S ribosomal RNA) markers were obtained for a subset of these samples. For each marker, genetic divergence within each species was in the same range as between species, confirming the close relationship between both species. Moreover, for each of the gene fragments, both species shared at least one haplotype/genotype. Hence, none of the molecular markers tested could be used, alone or in combination, to discriminate between L. illustris and L. caesar.

Belgian canine population and purebred study for forensics by improved mitochondrial DNA sequencing.

In canine population studies for forensics, the mitochondrial DNA is profiled by sequencing the two hyper variable regions, HV1 and HV2 of the control region. In a first effort to create a Belgian population database some samples showed partially poor sequence quality. We demonstrated that a nuclear pseudogene was co-amplified with the mtDNA control region. Using a new combination of primers this adverse result was no longer observed and sequencing quality was improved. All former samples with poor sequence data were reanalyzed. Furthermore, the forensic canine population study was extended to 208 breed and mixed dogs. In total, 58 haplotypes were identified, resulting in an exclusion capacity of 0.92. The profile distribution of the Belgian population sample was not significantly different from those observed in population studies of three other countries. In addition to the total population study 107 Belgian registered pedigree dogs of six breeds were profiled. Per breed, the obtained haplotypes were supplemented with those from population and purebred studies. The combined data revealed that some haplotypes were more or less prominent present in particular dog breeds. The statistically significant differences in haplotype distribution between breeds and population sample can have consequences on mtDNA databasing and matching probabilities in forensics.

COI sequence variability between Chrysomyinae of forensic interest.

About 50 Chrysomyinae specimens belonging to three forensic relevant species (Chrysomia albiceps, Phormia regina and Protophormia terraenovae) were collected from different geographical locations in Belgium over the last 5 yr. A 304-bp fragment of their mitochondrial COI gene is sequenced. The monophyletic branches of the phylogenetic tree reveal that this marker is suitable for discrimination between these species. The intra versus interspecific variability marks clear threshold levels for DNA barcoding. Nineteen Chrysomyinae specimens, collected from four locations in France, show mitotypes that are identical or at least very similar to the Belgian mitotypes. Considering additional specimens from outside of Europe reveals no intraspecific geographical variation within C. albiceps and P. terraenovae, whereas P. regina is subbranched in a Belgian-French and a USA-Chinese population.