mtDNA Typing Research Articles

Quantitative color analysis of burned bone to predict DNA quantity, quality, and genotyping success.

Badly burned skeletal remains are commonly submitted to forensic laboratories for victim identification via DNA analysis methods. Burned skeletal remains present many challenges for DNA analysis as they can contain low amounts of DNA which can also be damaged and degraded, resulting in partial or no STR profiles. Therefore, a simple, but effective screening method that identifies which samples may provide the most successful STR or mtDNA typing results for identification would enable forensic laboratories to save time, money, and resources. One metric that can be used and a screening method is the color of burned bone, as bone color changes with exposure to fire as temperature and length of exposure increase. This research developed a quantitative screening method based on the surface color of burned bone. The different visual bone colors (light brown, dark brown, black, gray, and white) were quantified using the Commission on Illumination L*a*b color space. These values were then compared to DNA yield, STR, and mtDNA profile completeness to identify whether the L*a*b values can predict genotyping success. A Bayesian network was constructed to determine the probability of STR typing success, given a set of L*a*b values. Results demonstrated that samples with an a* value greater than or equal to one and b* value greater than eight (light brown and dark brown burned samples) were the most predictive of STR typing success and mtDNA typing success. A decision tree for processing burned bones was constructed based on the color value thresholds.

Haplotype Diversity of Mitochondrial DNA in the Jat Population of Haryana

Abstract Background: The genetic representation of various population groups has been successfully attempted by studying hypervariable regions of human mitochondrial DNA (mtDNA) for forensic purposes. In this view of the fact, there are very few studies in the northern region of India focusing on mtDNA variations. The usage of mtDNA typing for forensic purposes has however been delayed in most states, especially Haryana, due to lack of mtDNA pool database for Haryana populations. Aims and Objectives: Since, there has been no elaborated mtDNA study on Jat population of Haryana, thus, the genetic variability of Jat population of Haryana was estimated by examining the hypervariable regions (HVI, HVII, and HVIII) of mtDNA. Materials and Methods: Blood samples of maternally unrelated individuals have been collected. Statistical Analysis Used: Different parameters and molecular diversity indices were computed. Results: The polymorphism has been observed with their corresponding haplogroups. The phylogenetic relationship was inferred between different haplogroups. Conclusion: A high level of variation and a low random match probability was observed in all the three hypervariable regions of mtDNA, indicating its utility for forensic purposes.

Neither Donor nor Recipient Mitochondrial Haplotypes Are Associated with Unrelated Donor Transplant Outcomes: A Validation Study from the CIBMTR

Graft-versus-host-disease (GVHD) is a multistep process that involves T-cell recognition and priming toward alloantigen, expansion, acquisition of effector function, and repeated tissue injury, resulting in clinical manifestations of the disease. All of these processes have considerable metabolic demands and understanding the key role of mitochondria in cellular metabolism as it relates to GVHD has increased significantly. Mitochondrial DNA (mtDNA) haplotypes have been linked to functional differences in vitro, suggesting they have functional differences at an organismal level. We previously used mtDNA typing to assess the impact of mtDNA haplotypes on outcomes of ~400 allo-HCT patients. This pilot study identified uncommon mtDNA haplotypes potentially associated with inferior outcomes. We sought to validate pilot findings of associations between donor and recipient mitochondrial haplotypes and transplant outcome. We examined a cohort of 4143 donor-recipient pairs obtained from the Center for International Blood and Marrow Transplant Research. MtDNA was extracted from whole blood or peripheral blood mononuclear cells from donors and recipients and sequenced to discern haplotype. We used multiple regression analysis to examine the independent association of mtDNA haplotype with overall survival and grade III-IV acute GVHD (aGVHD) adjusting for known risk factors for poor transplant outcome. Neither recipient nor donor mtDNA haplotype reached groupwise significance for overall survival (P =.26 and .39, respectively) or grade III-IV aGVHD (P = .68 and.57, respectively). Adjustment for genomically determined ancestry in the subset of donor-recipient pairs for which this was available did not materially change results. We conclude that our original finding was due to chance in a small sample size and that there is essentially no evidence that mtDNA haplotype or haplotype mismatch contributes to risk of serious outcomes after allogeneic transplantation.

Methods for the analysis of mitochondrial DNA

AbstractThe use of mitochondrial DNA (mtDNA) profiling as a tool for forming a link between an individual and a crime sample is a well‐established forensic biology technique. Though nuclear DNA typing is considered the gold standard method, certain evidence types have limited nuclear DNA available, such as telogen hairs and ancient or degraded remains, for which mtDNA analysis may be required. Traditionally mtDNA typing has been carried out using Sanger type sequencing, which is labor, time, and cost intensive. This has restricted the analysis of the mitochondrial genome to the control region, limiting the discrimination power of the technique. The introduction of massively parallel sequencing (MPS) into forensic laboratories has brought about the ability to analyze the whole mitochondrial genome of forensically relevant samples. The use of MPS for mtDNA analysis has resulted in an increase in the range of methods available for analysis, including: midi‐ and mini‐sized amplification strategies for whole genome analysis of casework‐type samples; whole genome single nucleotide polymorphism (SNP) multiplexes; and DNA capture methods allowing the analysis of highly degraded remains. The resulting increase in discriminatory power and the continued progression of the technology and associated methods increase its appeal as a DNA analysis method and may facilitate its implementation as a routinely used forensic tool. Here, we give an overview of current laboratory practices for the analysis of mtDNA using MPS.This article is categorized under: Forensic Biology > Haploid Markers Forensic Biology > Forensic DNA Technologies

Mitochondrial DNA data analysis strategies that inform MPS-based forensic casework implementation

Massively parallel sequencing (MPS) of human mitochondrial control region or whole genome is now faster, easier and more informative than was possible with Sanger sequencing and capillary electrophoresis. This study focuses on the aspects of mtDNA data that are important to consider when implementing MPS-based mtDNA typing into casework operations. This includes evaluation of data from the ForenSeq mtDNA typing system that indicate how interpretation concepts integral to Sanger sequencing of mtDNA apply directly to MPS-based mtDNA typing. In addition, there are new interpretation considerations that may be helpful when determining standard operating procedures and casework interpretation guidelines. Sequencing data are presented that may assist with SOP determination relative to sensitivity assessments that may help inform interpretation criteria, including thresholds, relative to input DNA template concentration ranges, number of pooled samples for MiSeq FGx sequencing runs and downstream bioinformatic demultiplexing. Given the quantitative nature of digital MPS data, these limit of detection studies may also assist when laboratories wish to set criteria for heteroplasmy detection (within an individual) or for mixed DNA sample interpretation. Assessment of variables will be provided across sample types such as hairs and PCR/sequencing inhibition when typing skeletal and dental remains to assist with fundamental understanding of ForenSeq mtDNA typing system limitations as well as metrics that may assist when making data quality determinations.

Mitochondrial DNA in human identification: a review.

Mitochondrial DNA (mtDNA) presents several characteristics useful for forensic studies, especially related to the lack of recombination, to a high copy number, and to matrilineal inheritance. mtDNA typing based on sequences of the control region or full genomic sequences analysis is used to analyze a variety of forensic samples such as old bones, teeth and hair, as well as other biological samples where the DNA content is low. Evaluation and reporting of the results requires careful consideration of biological issues as well as other issues such as nomenclature and reference population databases. In this work we review mitochondrial DNA profiling methods used for human identification and present their use in the main cases of humanidentification focusing on the most relevant issues for forensics.

Oral gram-positive bacterial DNA-based identification of saliva from highly degraded samples

Analyzing degraded evidence is an important challenge in forensic casework. Saliva remaining at a crime scene may deteriorate, due to various factors, making it difficult to identify. This study aims to clarify the efficacy of oral gram-positive and -negative bacterial DNA-based identification of saliva for analyzing highly degraded samples. Saliva samples were subjected to three different degradation treatments (heat denaturation: 40–80 °C in wet conditions; microbial decomposition: 1–5 days in humid soil; and ultraviolet (UV) irradiation: 0.01–1 J/cm2). We compared saliva markers’ detectability from the degraded samples—oral gram-positive bacterial DNA (Streptococcus salivarius and Streptococcus oralis), oral gram-negative bacterial DNA (Veillonella atypica and Prevotella maculosa) and salivary α-amylase. Oral bacterial DNA was detected using a melting curve analysis following real-time PCR. The efficacy of short tandem repeats (STR) and mitochondrial DNA (mtDNA) analyses were also compared. All oral bacterial DNA were detected with specific melting peaks from the heat-denatured samples, while neither catalytic nor immunochromatographic tests detected salivary α-amylase from the heat (80 °C) samples. The gram-positive bacterial DNA (S. salivarius and S. oralis) was detected from the microbial degradation (1–5 days) samples. In contrast, the gram-negative bacterial DNA (V. atypica and P. maculosa) and salivary α-amylase were not detected from samples treated for more than two days. UV exposure made bacterial DNA-based saliva identification difficult in a dose–dependent manner; however, UV irradiation did not influence protein-based saliva tests using salivary α-amylase as an indicator. As a result of STR and mtDNA typing, partial or null STR profiles were generated from the severely degraded (microbial (2–5 days) and UV (0.1–1 J/cm2) degradation) samples, but full mtDNA profiles were obtained from all degraded samples. The forensic applicability of bacterial DNA test evaluated, using mock case samples, indicates that the oral gram-positive bacterial DNA was more resistant to degradation than the other markers. We conclude that the oral gram-positive bacterial DNA-based examination could be useful for identifying saliva from severely environmentally-exposed forensic samples as well as mtDNA typing.

Estimation and Preparation of the Hypervariable Regions I/II Templates for Mitochondrial DNA Typing From Human Bones and Teeth Remains Using Singleplex Quantitative Polymerase Chain Reaction.

A method was designed for estimating and sequencing of mitochondrial DNA (mtDNA) that effectively and more quickly provides a complete mtDNA profile. In this context, we have developed this novel strategy for typing mtDNA from 10 bones and teeth remains (3 months to 44 years). The quantification of mtDNA was achieved by singleplex real-time polymerase chain reaction of the hypervariable region I fragment (445 bp) and hypervariable region II fragment (617 bp). Combined with the melting curve analysis, we have determined as little as 10 pg of mtDNA template that is suitable for sequence analysis. Furthermore, quantitative polymerase chain reaction products were directly used for following step of mtDNA typing by Sanger sequencing. This method allows the profile to be completely provided for faster human identification.

The influence of high temperature on the possibility of DNA typing in various human tissues.

The identification of unknown victims of high temperatures (fire, terrorist attack, and other disasters) is one of the most difficult tasks faced by forensic geneticists. The main aim of this study was to in­vestigate the availability of DNA isolated from various human tissue samples exposed to high temperatures of 100–1000°C for 5 and 10 minutes. Samples of varying thickness of thigh muscle, liver, heart, adipose tissue, bone, teeth, hair and nails of 52 fresh cadavers and 59 healthy teeth of 29 volunteers were used. The study was performed using the following commercially available STR (Short Tandem Repeats) and miniSTR kits: AmpFlSTR®SGM Plus® and AmpFlSTR®MiniFilerTM. Hyper variable region I (HVI) of human mitochondrial DNA (mtDNA) was sequenced with BigDye Terminator Cycle Sequencing Kit 1.1. The PEP (Primer-Extension Preamplification) method was used for the whole human genome amplification. It was possible to obtain complete DNA profiles (AmpFlSTR®SGM Plus®, AmpFlSTR®MiniFilerTM Applied Biosystems, USA and mtDNA HVI region) for tissue samples of heart, liver and thigh muscle, exposed up to 900°C for 5 min. However, under the applied conditions, limited usefulness of hair, nails and teeth for identification purposes was shown. DNA stability in tissues subjected to incineration depends on many factors, like tissue type and its thickness, temperature and time of exposure. In the cases of human remains exposed to high temperatures, samples of soft tissues of the highest weight (thickness) provide the best chance of successful identification through the genetic analysis. In some cases of negative results, even if using mtDNA typing, application of the whole genome amplification (WGA) technique could provide the expected results for highly degraded DNA templates.

Genetic identification with heteroplasmic variations in maternally relatedindividuals in forensic cases

Mitochondrial DNA (mtDNA), inherited from the mother, is used in a large variety of areas. In order to evaluate the connection between an incident and the victim or suspect in the analysis of criminal cases, DNA was obtained from buccal swabs sampled from 21 volunteers related through the mother's generation. Using Seqscape (v2.5.5) in an ABI 3100x1 capillary electrophoresis system, mtDNA sequence analyses were performed and compared to the revised Cambridge Reference Sequence. Individual mtDNA variations were detected. In three individuals, point heteroplasmy was observed. The results were evaluated according to the mtDNA typing guidelines established by the International Society for Forensic Genetics DNA Commission. This study can play an important role as a guide in cases of genomic DNA degradation or DNA absence, such as in disaster victim identification, where the genetic identification of an individual is necessary.

Evaluation of mtDNA stability across the maternal line: A study on three generations in two families

Sometimes deficiency paternity testing can not be resolved with routine STR analysis and nuclear DNA fails to give conclusive results. In these cases, mitochondrial DNA (mtDNA) analyses have been proved more effective. In general, mtDNA transmission is stable across many generations; therefore the mtDNA typing allows to assess maternal relationships, even in deficiency cases. To evaluate the consistency of mtDNA, we compared HVI and HVII regions in three generations along the maternal line (great-grandmother, grandmother, mother, daughter or son) in two families, confirming the useful of this marker in such cases.

Genetic diversity between two Igbo men from Owerri senatorial province as determined by autosomal short tandem repeats, Y-chromosomal short tandem repeats and mitochondrial DNA typing methods

Background: Human identification has recently been optimized following the completion of human genome sequence, by using DNA markers that exhibit the highest possible variation. However, in developing countries such as Nigeria, the application of DNA typing for identification of human subjects either for forensic or medical purposes is inadequate due to the absence of national forensic DNA laboratories and lack of the legislative framework. Materials and Methods: In this study, two male subjects of Igbo origin provided their blood, hair and buccal samples for DNA analysis. DNA was extracted, purified, quantified with human quantifiler™ polymerase chain reaction reaction mix. Hyper-variable segment 1 of the D-loop mitochondrial DNA (mtDNA HVS-1) was amplified, purified and fragments sequenced and analyzed with ABI Genetic Analyzer. A multiplex AmpFlSTR Identifiler kit amplified 15 STR plus amelogenin loci of the nuclear DNA and 16 STR of the Y-chromosome. GeneMapper ID software version 3.2 was used for the analysis of autosomal and Y-chromosome AmpFlSTR. Results: Result show that the mtDNA lineage of UMBACK subject belongs to L3e2b while ELAMBIA is assigned to L3f1b1 haplogroup. Based on the allelic frequency database, both subjects displayed uniqueness in random match probability for the autosomal allelic short-tandem repeat (STR). Based upon a mutation rate of 0.003 for Y-DNA STR markers, the two individuals most likely shared a common paternal ancestor 96 generations ago. Both subjects were assigned to E1b1a haplogroup with a 100% probability, which is consistent with the haplogroup associated with Igbo people in Nigeria. Conclusion: As expected, well-established forensic genetic tools comprising of mtDNA, autosomal and Y-chromsomomal STR typing methods were all found to distinguish two selected Nigerian Igbo individuals with a very high power of discrimination.

Mitochondrial DNA-based genetic evaluation of autochthonous cattle breeds in Croatia

Genetic diversity and phylogenetic relationship of three Croatian autochthonous cattle breeds was analyzed using a sequence of the mtDNA D-loop region. Among Busha, Istrian, and Slavonian Syrmian Podolian cattle 146 unrelated animals were tested. The sequencing of 780 base pairs of the mtDNA D-loop region revealed 39 polymorphic sites representing 28 different haplotypes. The highest numbers of haplotypes were observed in the Busha population and the lowest in the population of Slavonian Syrmian Podolian cattle, while the highest level of sequence diversity within a population was observed in the Istrian cattle. Our results indicate a high level of mtDNA diversity in the populations of Busha and Istrian cattle and a low level of preserved diversity in the population of Slavonian Syrmian Podolian cattle. The sequence analysis showed substantial subdivision between the breeds (F<sub>ST</sub> = 0.1434), and a large fraction of variation within the breeds. Although the dominant haplotypes are classified as the T3 haplogroup, some of the haplotypes are classified as the rarer T2 and T5 haplogroups. Genetic information based on mtDNA typing has a great importance for the future conservation management and preservation of genetic diversity in autochthonous cattle breeds.  

High throughput whole mitochondrial genome sequencing by two platforms of massively parallel sequencing

BackgroundMitochondrial DNA (mtDNA) is a valuable genetic bio-marker that has been implicated as a prognostic/diagnosticindicator for a number of diseases [1-3] as well as forhuman identification where forensic biologic evidencecontains too little or no nuclear DNA, such as a hair shaftwithout root or a fingernail, or where a sample from a dis-tant maternal relative is the only possibility for comparison[4-6]. Sanger sequencing has been the gold standardmethod for mtDNA typing, but the methodology has lim-itations with throughput, scalability, speed, and resolution[7]. Massively parallel sequencing technology (MPS) pro-vides platforms for more comprehensive coverage of thegenome per sample analyzed than currently is possiblewith Sanger sequencing [8,9]. Moreover, a number of dif-ferent samples, which can be distinguished by barcoding,may be sequenced simultaneously. Two of the availablepersonal genome sequencers are the Ion Torrent PersonalGenome Machine (PGM™) (LifeTechnologies, San Fran-cisco, CA) and the MiSeq

Identification of mammalian species using the short and highly variable regions of mitochondrial DNA

The mitochondrial DNA (mtDNA) typing is useful for the species determination of degraded samples and the nucleotide diversity of target fragments across species is crucial for the discrimination. In this study, the short and highly polymorphic regions flanked by two conserved termini were sought by the sequence alignment of mtDNA across species and two target regions located at 12S rRNA gene were characterized. Two universal primer sets were developed that appear to be effective for a wide variety of mammalian species, even for domestic birds. The two target regions could be efficiently amplified using their universal primer sets on degraded samples and provide sufficient information for species determination. Therefore, the two short and highly variable target regions might provide a high discriminative capacity and should be suitable for the species determination of degraded samples.

DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification

BackgroundMitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome.MethodsWe used in-solution hybridisation-based DNA capture (using DNA capture probes prepared from modern human mtDNA) to recover mtDNA from post-mortem human remains in which the majority of DNA is both highly fragmented (<100 base pairs in length) and chemically damaged. The method ‘immortalises’ the finite quantities of DNA in valuable extracts as DNA libraries, which is followed by the targeted enrichment of endogenous mtDNA sequences and characterisation by next-generation sequencing (NGS).ResultsWe sequenced whole mitochondrial genomes for human identification from samples where standard nuclear STR typing produced only partial profiles or demonstrably failed and/or where standard mtDNA hypervariable region sequences lacked resolving power. Multiple rounds of enrichment can substantially improve coverage and sequencing depth of mtDNA genomes from highly degraded samples. The application of this method has led to the reliable mitochondrial sequencing of human skeletal remains from unidentified World War Two (WWII) casualties approximately 70 years old and from archaeological remains (up to 2,500 years old).ConclusionsThis approach has potential applications in forensic science, historical human identification cases, archived medical samples, kinship analysis and population studies. In particular the methodology can be applied to any case, involving human or non-human species, where whole mitochondrial genome sequences are required to provide the highest level of maternal lineage discrimination. Multiple rounds of in-solution hybridisation-based DNA capture can retrieve whole mitochondrial genome sequences from even the most challenging samples.

Mitochondrial DNA polymorphisms in Phytophthora infestans: New haplotypes are identified and re-defined by PCR

Polymorphisms of mitochondrial DNA (mt-DNA) are particularly useful for monitoring specific pathogen populations like Phytophthora infestans. Basically type I and II of P. infestans mt-DNA were categorized by means of polymorphism lengths caused by an ~2kb insertion, which can be detected via restriction enzyme digestion. In addition genome sequencing of haplotype Ib has been used as a simple Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR–RFLP) method to indirectly identify type I and II alterations through EcoR I restriction enzyme DNA fragment patterns of the genomic P4 area. However, with the common method, wrong mt-DNA typing occurs due to an EcoR I recognition site mutation in the P4 genomic area. Genome sequencing of the four haplotypes (Ia, Ib, IIa, and IIb) allowed us to thoroughly examine mt-DNA polymorphisms and we indentified two hypervariable regions (HVRs) named HVRi and HVRii. The HVRi length polymorphism caused by a 2kb insertion/deletion was utilized to identify mt-DNA types I and II, while another length polymorphism in the HVRii region is caused by a variable number of tandem repeats (n=1, 2, or 3) of a 36bp sized DNA stretch and was further used to determine mt-DNA sub-types, which were described as Rn=1, 2, or 3. Finally, the P. infestans mt-DNA haplotypes were re-defined as IR1 or IIR2 according to PCR derived HVRi and HVRii length polymorphisms. Twenty-three isolates were chosen to verify the feasibility of our new approach for identifying mt-DNA haplotypes and a total of five haplotypes (IR1, IR2, IR3, IIR2 and IIR3) were identified. Additionally, we found that six isolates determined as type I by our method were mistakenly identified as type II by the PCR–RFLP technique. In conclusion, we propose a simple and rapid PCR method for identification of mt-DNA haplotypes based on sequence analyses of the mitochondrial P. infestans genome.

New guidelines for the publication of genetic population data

New guidelines for the publication of genetic population data

MtDNA typing of single-sperm cells isolated by micromanipulation

Some sexual assault crimes constitute a problem for the legal institutions confronted with the DNA analysis of such cases. Often, sperm cells are found in the victim's vaginal tract during medical examination but their successful genotyping is compromised by the huge excess of the victim's epithelial cells as well as by the degradation of genomic DNA present in sperm cells as a consequence of female immune response.Mitochondrial DNA present in the mid-piece of sperm cells might be useful in some specific cases in order to differentiate the donors of a semen sample. The high number of copies per cell and its circular nature that may confer some protection from the action of exonucleases make it more suitable for cases where few cells are available and/or the DNA is degraded.We have developed a novel strategy for typing mtDNA from single-sperm cells. Specific amplification of male mitochondrial DNA is ensured by use of sequence specific primers designed on the basis of mitochondrial single nucleotide polymorphisms existent throughout the control region. The strategy was applied to single-sperm cells isolated by micromanipulation from slides smeared with vaginal swabs taken immediately after sexual intercourse of voluntary couples. After sequencing the PCR products, it was possible to obtain a match between the DNA sequence from the buccal swab and the DNA sequence of the single sperm-cell, for each voluntary man. With this new strategy, the problem of contamination with DNA from the victim observed when using universal primers was completely overtaken. This method will probably allow the resolution of multiple-rapist crimes, where the collected sperm cells can be separately typed.

MtGenome reference population databases and the future of forensic mtDNA analysis

Mitochondrial DNA (mtDNA) testing in the forensic context requires appropriate, high quality population databases for estimating the rarity of questioned haplotypes. Currently, however, available forensic mtDNA reference databases only include information from the mtDNA control region. While this information is obviously strengthening the foundation upon which current mtDNA identification efforts are based, these data do not adequately prepare the field for recent and rapid advancements in mtDNA typing technologies. Novel tools that quickly and easily permit access to mtDNA coding region data for increased discrimination are now available in the form of single nucleotide polymorphism assays, sequence specific oligonucleotide probes, mass spectrometry instrumentation and next generation sequencing technologies. However, the randomly sampled entire mtGenome reference population data required for statistical interpretation of coding region data are lacking. As a result, in the near future, it seems that routine use of mtDNA coding region data in forensic case work will depend more upon the availability of high-quality entire mtGenome population reference data than the ease with which coding region data can be generated from evidence specimens. Until mtGenome reference databases are available, the utility of novel mtDNA typing technologies and the benefits of recovering mtDNA coding region information from forensic specimens will be limited. Thus, future mtDNA databasing efforts are needed for the development of entire mtDNA genome reference population data suitable for forensic comparisons.