Publications by authors named "Thomas J Parsons"

46 Publications

Evidence for multi-copy Mega-NUMTs in the human genome.

Nucleic Acids Res 2021 02;49(3):1517-1531

Institute of Legal Medicine, Medical University of Innsbruck, Innsbruck 6020, Austria.

The maternal mode of mitochondrial DNA (mtDNA) inheritance is central to human genetics. Recently, evidence for bi-parental inheritance of mtDNA was claimed for individuals of three pedigrees that suffered mitochondrial disorders. We sequenced mtDNA using both direct Sanger and Massively Parallel Sequencing in several tissues of eleven maternally related and other affiliated healthy individuals of a family pedigree and observed mixed mitotypes in eight individuals. Cells without nuclear DNA, i.e. thrombocytes and hair shafts, only showed the mitotype of haplogroup (hg) V. Skin biopsies were prepared to generate ρ° cells void of mtDNA, sequencing of which resulted in a hg U4c1 mitotype. The position of the Mega-NUMT sequence was determined by fluorescence in situ hybridization and two different quantitative PCR assays were used to determine the number of contributing mtDNA copies. Thus, evidence for the presence of repetitive, full mitogenome Mega-NUMTs matching haplogroup U4c1 in various tissues of eight maternally related individuals was provided. Multi-copy Mega-NUMTs mimic mixtures of mtDNA that cannot be experimentally avoided and thus may appear in diverse fields of mtDNA research and diagnostics. We demonstrate that hair shaft mtDNA sequencing provides a simple but reliable approach to exclude NUMTs as source of misleading results.
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http://dx.doi.org/10.1093/nar/gkaa1271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7897518PMC
February 2021

Challenges with co-amplification of microbial DNA in interpretation of STR profiles obtained from human skeletal remains.

Forensic Sci Int Genet 2021 Mar 9;51:102452. Epub 2021 Jan 9.

International Commission on Missing Persons, Koninginnegracht 12, Den Haag, 2514 AA, the Netherlands. Electronic address:

STR-based DNA analysis is still the main tool for human DNA identification in most forensic DNA laboratories. DNA typing of aged human skeletal elements faces well-known interpretation challenges characteristic of degraded and low copy number DNA samples. Analyzing tens of thousands of human bone and teeth samples, we found that the occasional presence of artefactual peaks of presumed microbial origin adds another layer of complexity to the interpretation of STR profiles. In this paper, we present our approach and suggest guidelines for identifying and distinguishing non-human peaks, developed over the last 18 years. Additionally, we report a compendium of artefact peaks of presumed microbial origin recorded in human STR profiles obtained from bone and teeth samples, originating from Iraq, Chile, Maldives, Brazil and Western Balkans. Our experience has shown that these artefacts are not uncommon in bone STR testing, suggesting the possibility of occurrence in other forensic contexts, particularly trace DNA samples. Raising awareness among the forensic DNA community and accounting for this phenomenon is important for accurate STR interpretation.
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http://dx.doi.org/10.1016/j.fsigen.2020.102452DOI Listing
March 2021

Impact of DNA degradation on massively parallel sequencing-based autosomal STR, iiSNP, and mitochondrial DNA typing systems.

Int J Legal Med 2019 Sep 2;133(5):1369-1380. Epub 2019 Jul 2.

Department of Biochemistry and Molecular Biology, Forensic Science Program, Pennsylvania State University, State College, PA, USA.

Biological samples, including skeletal remains exposed to environmental insults for extended periods of time, exhibit increasing levels of DNA damage and fragmentation. Human forensic identification methods typically use a combination of mitochondrial (mt) DNA sequencing and short tandem repeat (STR) analysis, which target segments of DNA ranging from 80 to 500 base pairs (bps). Larger templates are often unavailable as skeletal samples age and the associated DNA degrades. Single-nucleotide polymorphism (SNP) loci target shorter templates and may serve as a solution to the problem. Recently developed assays for STR and SNP analysis using a massively parallel sequencing approach, such as the ForenSeq kit (Verogen, San Diego, CA), offer a means for generating results from degraded samples as they target templates down to 60 to 170 bps. We performed a modeling study that demonstrates that SNPs can increase the significance of an identification when analyzing DNA down to an average size of 100 bps for input amounts between 0.375 and 1 ng of nuclear DNA. Observations from this study were then compared with human skeletal material results (n = 14, ninth to eighteenth centuries), which further demonstrated the utility of the ForenSeq kit for degraded samples. The robustness of the Promega PowerSeq™ Mito System was also tested with human skeletal remains (n = 70, ninth to eighteenth centuries), resulting in successful coverage of 99.29% of the mtDNA control region at 50× coverage or more. This was accompanied by modifications to a mainstream DNA extraction technique for skeletal remains that improved recovery of shorter templates.
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http://dx.doi.org/10.1007/s00414-019-02110-4DOI Listing
September 2019

Large scale DNA identification: The ICMP experience.

Forensic Sci Int Genet 2019 01 15;38:236-244. Epub 2018 Nov 15.

International Commission on Missing Persons, Koninginnegracht 12a, The Hague, Netherlands.

The International Commission on Missing Persons (ICMP) is a treaty-based international organization with a global mandate to address the issue of missing persons. It works with governments, civil society organizations, and others, and utilizes data systems and technical assistance in forensic science. ICMP's initial work focused on the ∼40,000 people missing in the Western Balkans from the conflicts of the 1990s. A "DNA-led" approach to large-scale DNA identification of the missing was developed, based on high-throughput autosomal STR testing of skeletal remains from mass graves and other sites, and the establishment of a regional database of DNA profiles from family members of the missing. Database pairwise and pedigree kinship searching is conducted using in-house DNA matching software, the Identification Data Management System (iDMS), providing high-certainty DNA matches that are integrated in a multi-disciplinary identification process. Anthropological guidelines for sampling skeletal remains for DNA testing are based on tens of thousands of tests from a wide range of skeletal elements, allowing for prioritization based on DNA preservation. Large-scale collection of family reference samples has been conducted, resulting in a database of more than 100,000 family reference DNA profiles across all projects and delivering family DNA match reports for more than 20,000 individuals. From the 1995 Srebrenica event, ICMP provided DNA matches for 6887 of the ∼8000 missing from that event. In assistance to justice, ICMP has provided extensive evidence and expert testimony in multiple war crimes trials, including those conducted at the ICTY. This article provides an overview of ICMP's technical involvement over the last 17 years in areas of DNA testing and database matching, and training and capacity building projects with partners. It also touches on the development of massively parallel sequencing (MPS) strategies specifically tailored to missing persons applications.
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http://dx.doi.org/10.1016/j.fsigen.2018.11.008DOI Listing
January 2019

An economical and efficient method for postmortem DNA sampling in mass fatalities.

Forensic Sci Int Genet 2018 09 10;36:167-175. Epub 2018 Jul 10.

International Commission on Missing Persons Headquarters, Koninginnegracht 12 2514 AA Den Haag, The Netherlands.

In mass fatality events, the need to identify large numbers of deceased persons using DNA can be a significant drain on already overburdened forensic practitioners, both in the field setting and the laboratory. The laboratory may be required to extract DNA from a variety of postmortem sample types, family or direct reference samples related to the missing, and perform matching of these results in a short period of time. While most forensic institutions are well equipped to handle both family and direct reference samples, postmortem samples such as bone or heterogeneous tissue samples can be difficult for labs to analyze. We have devised an easily deployable, efficient, and inexpensive method for collecting postmortem DNA samples on commercially available DNA preservation cards ("FTA" cards). FTA cards are already widely used in forensic labs and are convenient for shipping due to their small volume and stability at room temperature. We evaluated the suitability of a protocol involving swabbing of incisions made on cadavers and sample deposition onto FTA cards over various postmortem intervals and under different environmental conditions. Each trial took place during a different point in the calendar year to evaluate the effects of seasonal weather patterns and temperature on decomposition, DNA yield, and rates of DNA degradation. To further account for the effects of seasonality (temperature and humidity), the progression of body decomposition was recorded following the Total Body Score (TBS) method [1]. DNA degradation was assessed either through STR amplification of 1.2 mm FTA punches or DNA extraction from 3.0 mm punches followed by real-time PCR quantification and STR amplification and genotyping. No consistent relationship was observed between postmortem interval and DNA degradation. Instead, the TBS score, which captures the stage of body decomposition, was shown to correlate well with DNA quantity. A TBS of 15 and below consistently yielded strong partial or full profiles (20 STR loci and Amelogenin using the PowerPlex 21 System) from all individuals from either 1.2 mm or 3.0 mm punches. Transfer of sample swabs to FTA cards is shown to be a simple and effective method for both field and laboratory operations over a range of conditions that can be evaluated by field forensic practitioners based on a body decomposition score. The approach could be beneficially integrated into mass fatality response plans.
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http://dx.doi.org/10.1016/j.fsigen.2018.07.009DOI Listing
September 2018

Identification of Missing Norwegian World War II Soldiers, in Karelia Russia.

J Forensic Sci 2015 Jul 24;60(4):1104-10. Epub 2015 Mar 24.

The International Commission on Missing Persons, 71000, Sarajevo, Bosnia and Herzegovina.

This article presents the multidisciplinary effort in trying to identify the skeletal remains of 100 Norwegian soldiers serving in the German army, killed in Karelia Russia in 1944, from the recovery of the remains through the final identification using DNA. Of the 150 bone samples sent for DNA testing, 93 DNA profiles were obtained relating to 57 unique individuals. The relatives could not be directly contacted as the soldiers were considered as traitors to Norway; therefore, only 45 reference samples, relating to 42 cases of the missing, were donated. DNA matches for 14 soldiers and 12 additional body part re-associations for these individuals were found. Another 24 bone samples were re-associated with 16 individuals, but no familial match was found. More than six decades after the end of WWII, DNA analysis can significantly contribute to the identification of the remains.
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http://dx.doi.org/10.1111/1556-4029.12767DOI Listing
July 2015

Characterization of mitochondrial DNA control region lineages in Iraq.

Int J Legal Med 2013 Mar 29;127(2):373-5. Epub 2012 Aug 29.

Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Via Ferrata 1, 27100, Pavia, Italy.

To evaluate the utility of mtDNA control region data for the purposes of forensic DNA testing in Iraq, a sample of 182 subjects (128 Arab Muslims, 15 Kurd Muslims, 22 Assyrian Christians and 17 Mandaean Arabs) was tested. High numbers of singleton haplotypes were observed among Arabs, Kurds and Assyrians, but fewer were found in Mandaeans. High molecular diversity and low random match probabilities confirmed the value of control region data in the investigation of maternal genetic lineages among the Iraqi population.
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http://dx.doi.org/10.1007/s00414-012-0757-8DOI Listing
March 2013

Characterization of a modified amplification approach for improved STR recovery from severely degraded skeletal elements.

Forensic Sci Int Genet 2012 Sep 7;6(5):578-87. Epub 2012 Mar 7.

Armed Forces DNA Identification Laboratory, 1413 Research Blvd., Rockville, MD 20850, USA.

Degraded skeletal remains generally contain limited quantities of genetic material and thus DNA-based identification efforts often target the mitochondrial DNA (mtDNA) control region due to the relative abundance of intact mtDNA as compared to nuclear DNA. In many missing person cases, however, the discriminatory power of mtDNA is inadequate to permit identification when associated anthropological, odontological, or contextual evidence is also limited, and/or the event involves a large number of individuals. In situations such as these, more aggressive amplification protocols which can permit recovery of STR data are badly needed as they may represent the last hope for conclusive identification. We have previously demonstrated the potential of a modified Promega PowerPlex 16 amplification strategy for the recovery of autosomal STR data from severely degraded skeletal elements. Here, we further characterize the results obtained under these modified parameters on a variety of sample types including pristine control DNA and representative case work specimens. Not only is the amplification approach evaluated here sensitive to extremely low authentic DNA input quantities (6 pg), but when the method was applied to thirty-one challenging casework specimens, nine or more alleles were reproducibly recovered from 69% of the samples tested. Moreover, when we independently considered bone samples extracted with a protocol that includes complete demineralization of the bone matrix, the percentage of samples yielding nine or more reproducible alleles increased to 95% with the modified amplification parameters. Overall, direct comparisons between the modified amplification protocol and the standard amplification protocol demonstrated that allele recovery was significantly greater using the aggressive parameters, with only a minimal associated increase in artifactual data.
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http://dx.doi.org/10.1016/j.fsigen.2012.01.010DOI Listing
September 2012

DNA extraction from aged skeletal samples for STR typing by capillary electrophoresis.

Methods Mol Biol 2012 ;830:185-98

International Commission on Missing Persons, Sarajevo, Bosnia and Herzegovina.

STR analysis of DNA extracted from skeletal samples can play an important role in the identification of missing persons. Here we present a method for the extraction of DNA from skeletal samples involving complete demineralization and digestion of the sample, followed by purification by silica binding. This method, together with the multiplex STR typing approach also presented, has proven highly successful in the recovery of DNA profiles from degraded, aged skeletal remains from a wide range of environmental contexts. The methodological steps presented include bone decontamination and grinding, DNA extraction, repurification in the case of highly inhibited samples, quantification, STR multiplex amplification, and profile reporting guidelines. However, the conditions applied for amplification and the criteria for allele calling and profile submission must be based on the results of each laboratory's internal validation experiments involving the type of samples relevant to the project at hand. The methods presented here have permitted large-scale DNA-based identification of persons missing from mass disasters and armed conflict.
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http://dx.doi.org/10.1007/978-1-61779-461-2_13DOI Listing
March 2012

Automatable full demineralization DNA extraction procedure from degraded skeletal remains.

Forensic Sci Int Genet 2012 May 31;6(3):398-406. Epub 2011 Aug 31.

International Commission on Missing Persons, Alipasina 45a, Sarajevo, Bosnia and Herzegovina.

During the 7 year period from 2002 to 2009 a high volume, silica-binding DNA extraction protocol for bone, based on modified QIAGEN's Blood Maxi Kit protocol was highly successful permitting the DNA matching of >14,500 missing persons from former Yugoslavia. This method, however, requires large amount of bone material and large volumes of reagents. The logical evolution was to develop a more efficient extraction protocol for bone samples that uses significantly less starting material while increasing the success in obtaining DNA results from smaller, more challenging samples. In this study we compared the performance of ICMP's original protocol against an automatable full demineralization approach. In order to provide reliable results and to simulate a wide variety of cases, we analyzed 40 bone samples in a comparative study based on DNA concentrations and quality of resulting STR profiles. The new protocol results in the dissolution of the entire bone powder sample, thus eliminating the possibility that DNA is left behind, locked in remaining solid bone matrix. For the majority of samples tested, the DNA concentrations obtained from half a gram of fully digested bone material were equivalent to or greater than the ones obtained from 2g of partially demineralized bone powder. Furthermore, the full demineralization process significantly increases the proportion of full profiles reflecting the correlation with better DNA quality. This method has been adapted for the QIAcube robotic platform. The performance of this automated full demineralization protocol is similar to the manual version and increases overall lab throughput. It also simplifies the process by eliminating quality control procedures that are advisable in manual procedures, and overall reduces the chance of human error. Finally we described a simple and efficient post-extraction clean-up method that can be applied to DNA extracts obtained from different protocols. This protocol has also been adjusted for the QIAcube platform.
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http://dx.doi.org/10.1016/j.fsigen.2011.08.004DOI Listing
May 2012

Assessing a novel room temperature DNA storage medium for forensic biological samples.

Forensic Sci Int Genet 2012 Jan 15;6(1):31-40. Epub 2011 Feb 15.

Forensic Science Program, Justice Studies Department, MacQuarrie Hall 521, San Jose State University, San Jose, CA 95192, USA.

The ability to properly collect, analyze and preserve biological stains is important to preserving the integrity of forensic evidence. Stabilization of intact biological evidence in cells and the DNA extracts from them is particularly important since testing is generally not performed immediately following collection. Furthermore, retesting of stored DNA samples may be needed in casework for replicate testing, confirmation of results, and to accommodate future testing with new technologies. A novel room temperature DNA storage medium, SampleMatrix™ (SM; Biomatrica, Inc., San Diego, CA), was evaluated for stabilizing and protecting samples. Human genomic DNA samples at varying amounts (0.0625-200 ng) were stored dry in SM for 1 day to 1 year under varying conditions that included a typical ambient laboratory environment and also through successive freeze-thaw cycles (3 cycles). In addition, spiking of 1-4 × SM into samples prior to analysis was performed to determine any inhibitory effects of SM. Quantification of recovered DNA following storage was determined by quantitative PCR or by agarose gel electrophoresis, and evaluation of quantitative peak height results from multiplex short tandem repeat (STR) analyses were performed to assess the efficacy of SM for preserving DNA. Results indicate no substantial differences between the quality of samples stored frozen in liquid and those samples maintained dry at ambient temperatures protected in SM. For long-term storage and the storage of low concentration samples, SM provided a significant advantage over freezer storage through higher DNA recovery. No detectable inhibition of amplification was observed at the recommended SM concentration and complete profiles were obtained from genomic DNA samples even in the presence of higher than recommended concentrations of the SM storage medium. The ability to stabilize and protect DNA from degradation at ambient temperatures for extended time periods could have tremendous impact in simplifying and improving sample storage conditions and requirements. The current work focuses on forensics analysis; however this technology is applicable to all endeavors requiring storage of DNA.
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http://dx.doi.org/10.1016/j.fsigen.2011.01.008DOI Listing
January 2012

The mtDNA composition of Uzbekistan: a microcosm of Central Asian patterns.

Int J Legal Med 2010 May 6;124(3):195-204. Epub 2010 Feb 6.

Research Department, Armed Forces DNA Identification Laboratory (AFDIL), 1413 Research Blvd, Rockville, MD, 20850, USA.

In order to better characterize and understand the mtDNA population genetics of Central Asia, the mtDNA control regions of over 1,500 individuals from Uzbekistan have been sequenced. Although all samples were obtained from individuals residing in Uzbekistan, individuals with direct ancestry from neighboring Central Asian countries are included. Individuals of Uzbek ancestry represent five distinct geographic regions of Uzbekistan: Fergana, Karakalpakstan, Khorezm, Qashkadarya, and Tashkent. Individuals with direct ancestry in nearby countries originate from Kazakhstan, Kyrgyzstan, Russia, Afghanistan, Turkmenistan, and Tajikistan. Our data reinforce the evidence of distinct clinal patterns that have been described among Central Asian populations with classical, mtDNA, and Y-chromosomal markers. Our data also reveal hallmarks of recent demographic events. Despite their current close geographic proximity, the populations with ancestry in neighboring countries show little sign of admixture and retain the primary mtDNA patterns of their source populations. The genetic distances and haplogroup distributions among the ethnic populations are more indicative of a broad east-west cline among their source populations than of their relatively small geographic distances from one another in Uzbekistan. Given the significant mtDNA heterogeneity detected, our results emphasize the need for heightened caution in the forensic interpretation of mtDNA data in regions as historically rich and genetically diverse as Central Asia.
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http://dx.doi.org/10.1007/s00414-009-0406-zDOI Listing
May 2010

Mitochondrial control region sequences from an African American population sample.

Forensic Sci Int Genet 2009 Dec 31;4(1):e45-52. Epub 2009 May 31.

Armed Forces DNA Identification Laboratory, 1413 Research Boulevard, Building 101, Rockville, MD 20850, USA.

Entire mitochondrial control region data were generated for 248 African American individuals, which had been previously typed for 15 autosomal STRs [J.M. Butler, R. Schoske, P.M. Vallone, J.W. Redman, M.C. Kline, Allele frequencies for 15 autosomal STR loci on U.S. Caucasian, African American, and Hispanic populations, J. Forensic Sci. 48 (2003) 908-911].
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http://dx.doi.org/10.1016/j.fsigen.2009.04.010DOI Listing
December 2009

Mitochondrial DNA control region variation in a population sample from Hong Kong, China.

Forensic Sci Int Genet 2009 Sep 11;3(4):e119-25. Epub 2008 Dec 11.

Armed Forces DNA Identification Laboratory, Rockville, MD 20850, United States.

Entire mitochondrial control region sequences were generated from 377 unrelated individuals from urban Hong Kong. In line with other control region datasets from China, the sample from Hong Kong exhibited significant genetic diversity that was reflected in a random match probability of 0.19% and a mean pairwise difference of 13.14. A total of 305 haplotypes were identified, of which 262 were unique. These sequences will be made publicly available to serve as forensic mtDNA reference data for China.
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http://dx.doi.org/10.1016/j.fsigen.2008.10.008DOI Listing
September 2009

The use of mitochondrial DNA single nucleotide polymorphisms to assist in the resolution of three challenging forensic cases.

J Forensic Sci 2009 Jul 26;54(4):887-91. Epub 2009 May 26.

Armed Forces DNA Identification Laboratory, Armed Forces Institute of Pathology, AFIP Annex, 1413 Research Blvd., Rockville, MD 20850, USA.

Mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) in an 11-plex assay were typed in three missing person cases involving highly degraded human remains. Unlike the traditional forensic approach to analyzing mtDNA which focuses on sequencing portions of the noncoding Control Region, this assay targets discriminatory SNPs that reside principally in the coding region. In two of the cases, the SNP typing successfully excluded one of two reference families that could not be excluded on the basis of mtDNA hypervariable region sequencing alone, and resulted in the final resolution of both decades-old cases. In a third case, SNP typing confirmed the sorting and reassociation of multiple commingled skeletal elements. The application of a specific mtDNA SNP assay in these cases demonstrates its utility in distinguishing samples when the most common Caucasian hypervariable region type is encountered in forensic casework.
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http://dx.doi.org/10.1111/j.1556-4029.2009.01069.xDOI Listing
July 2009

Mitochondrial control region sequences from an Egyptian population sample.

Forensic Sci Int Genet 2009 Jun 5;3(3):e97-103. Epub 2008 Nov 5.

Armed Forces DNA Identification Laboratory, 1413 Research Boulevard, Building 101, Rockville, MD 20850, USA.

Entire mitochondrial control region data was generated for 277 unrelated Egyptian individuals. High-throughput robotics, a redundant sequencing approach, and several quality control checks were implemented to generate a high-quality database. The data presented here will augment the limited Egyptian mtDNA reference data currently available for forensic comparisons.
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http://dx.doi.org/10.1016/j.fsigen.2008.09.004DOI Listing
June 2009

Investigation of heteroplasmy in the human mitochondrial DNA control region: a synthesis of observations from more than 5000 global population samples.

J Mol Evol 2009 May 1;68(5):516-27. Epub 2009 May 1.

Research Department, Armed Forces DNA Identification Laboratory, 1413 Research Blvd., Rockville, MD 20850, USA.

Instances of point and length heteroplasmy in the mitochondrial DNA control region were compiled and analyzed from over 5,000 global human population samples. These data represent observations from a large and broad population sample, representing nearly 20 global populations. As expected, length heteroplasmy was frequently observed in the HVI, HVII and HVIII C-stretches. Length heteroplasmy was also observed in the AC dinucleotide repeat region, as well as other locations. Point heteroplasmy was detected in approximately 6% of all samples, and while the vast majority of heteroplasmic samples comprised two molecules differing at a single position, samples exhibiting two and three mixed positions were also observed in this data set. In general, the sites at which heteroplasmy was most commonly observed correlated with reported control region mutational hotspots. However, for some sites, observations of heteroplasmy did not mirror established mutation rate data, suggesting the action of other mechanisms, both selective and neutral. Interestingly, these data indicate that the frequency of heteroplasmy differs between particular populations, perhaps reflecting variable mutation rates among different mtDNA lineages and/or artifacts of particular population groups. The results presented here contribute to our general understanding of mitochondrial DNA control region heteroplasmy and provide additional empirical information on the mechanisms contributing to mtDNA control region mutation and evolution.
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http://dx.doi.org/10.1007/s00239-009-9227-4DOI Listing
May 2009

Complete mitochondrial genome sequences for 265 African American and U.S. "Hispanic" individuals.

Forensic Sci Int Genet 2008 Jun 1;2(3):e45-8. Epub 2008 Feb 1.

Armed Forces DNA Identification Laboratory, 1413 Research Boulevard, Building 101, Rockville, MD 20850, USA.

Entire mitochondrial genome (mtGenome) sequences of 265 unrelated African American and U.S. "Hispanic" individuals were generated.
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http://dx.doi.org/10.1016/j.fsigen.2007.12.001DOI Listing
June 2008

Mitochondrial control region sequences from a U.S. "Hispanic" population sample.

Forensic Sci Int Genet 2008 Mar;2(2):e19-23

Armed Forces DNA Identification Laboratory, Rockville, MD 20850, United States.

Entire mitochondrial control region data was generated for 128 "Hispanics" from the United States. These samples have been previously typed for 15 autosomal STRs [J.M. Butler, R. Schoske, P.M. Vallone, J.W. Redman, M.C. Kline, Allele frequencies for 15 autosomal STR loci on U.S. Caucasian, African American, and Hispanic populations, J. Forensic Sci. 48 (2003) 908-911]. High-throughput robotics, a redundant sequencing approach, and several quality control checks were implemented to generate a high-quality database. The data presented here will augment Hispanic reference data available for forensic mtDNA comparisons.
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http://dx.doi.org/10.1016/j.fsigen.2007.11.004DOI Listing
March 2008

Mitochondrial control region sequences from a Vietnamese population sample.

Int J Legal Med 2008 May 25;122(3):257-9. Epub 2007 Oct 25.

Armed Forces DNA Identification Laboratory, 1413 Research Boulevard, Building 101, Rockville, MD 20850, USA.

Entire mitochondrial control region data were generated for 187 individuals from Vietnam. These samples have been previously typed for 16 autosomal short-tandem repeats (STRs) [1].
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http://dx.doi.org/10.1007/s00414-007-0205-3DOI Listing
May 2008

Application of low copy number STR typing to the identification of aged, degraded skeletal remains.

J Forensic Sci 2007 Nov 17;52(6):1322-7. Epub 2007 Oct 17.

Armed Forces DNA Identification Laboratory, Armed Forces Institute of Pathology, AFIP Annex, 1413 Research Blvd., Rockville, MD 20850, USA.

Low copy number (LCN) STR typing was successfully applied to four interesting cases during developmental validation of the approach for degraded skeletal remains. Specific questions were addressed in each case, with the acquisition of STR data largely serving as additional confirmatory or investigatory information in any specific situation, and not necessarily providing the definitive evidence to establish identity. The cases involve missing U.S. service members from World War I, World War II, and the Vietnam War. The variety of these cases, in terms of the questions addressed, the age of the remains, and the type of reference material available for comparison, demonstrates the broad utility of LCN STR typing in the identification of degraded skeletal remains from missing persons.
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http://dx.doi.org/10.1111/j.1556-4029.2007.00561.xDOI Listing
November 2007

Variant alleles, triallelic patterns, and point mutations observed in nuclear short tandem repeat typing of populations in Bosnia and Serbia.

Croat Med J 2007 Aug;48(4):494-502

International Commission on Missing Persons, Sarajevo, Bosnia and Herzegovina.

Aim: To present a compendium of off-ladder alleles and other genotyping irregularities relating to rare/unexpected population genetic variation, observed in a large short tandem repeat (STR) database from Bosnia and Serbia.

Methods: DNA was extracted from blood stain cards relating to reference samples from a population of 32800 individuals from Bosnia and Serbia, and typed using Promega's PowerPlex16 STR kit.

Results: There were 31 distinct off-ladder alleles were observed in 10 of the 15 STR loci amplified from the PowerPlex16 STR kit. Of these 31, 3 have not been previously reported. Furthermore, 16 instances of triallelic patterns were observed in 9 of the 15 loci. Primer binding site mismatches that affected amplification were observed in two loci, D5S818 and D8S1179.

Conclusion: Instances of deviations from manufacturer's allelic ladders should be expected and caution taken to properly designate the correct alleles in large DNA databases. Particular care should be taken in kinship matching or paternity cases as incorrect designation of any of these deviations from allelic ladders could lead to false exclusions.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2080559PMC
August 2007

Success rates of nuclear short tandem repeat typing from different skeletal elements.

Croat Med J 2007 Aug;48(4):486-93

International Commission on Missing Persons, Sarajevo, Bosnia and Herzegovina.

Aim: To evaluate trends in DNA typing success rates of different skeletal elements from mass graves originating from conflicts that occurred in the former Yugoslavia (Bosnia and Herzegovina and Kosovo) during the 1990s, and to establish correlation between skeletal sample age and success of high throughput short tandem repeat (STR) typing in the large data set of the International Commission on Missing Persons.

Method: DNA extraction and short tandem repeat (STR) typing have been attempted on over 25000 skeletal samples. The skeletal samples originated from different geographical locations where the conflicts occurred and from different time periods from 1992 to 1999. DNA preservation in these samples was highly variable, but was often significantly degraded and of limited quantity. For the purpose of this study, processed samples were categorized according to skeletal sample type, sample age since death, and success rates tabulated.

Results: Well-defined general trends in success rates of DNA analyses were observed with respect to the type of bone tested and sample age. The highest success rates were observed with samples from dense cortical bone of weight-bearing leg bones (femur 86.9%), whereas long bones of the arms showed significantly lower success (humerus 46.2%, radius 24.5%, ulna 22.8%). Intact teeth also exhibited high success rates (teeth 82.7%). DNA isolation from other skeletal elements differed considerably in success, making bone sample selection an important factor influencing success.

Conclusion: The success of DNA typing is related to the type of skeletal sample. By carefully evaluating skeletal material available for forensic DNA testing with regard to sample age and type of skeletal element available, it is possible to increase the success and efficiency of forensic DNA testing.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2080564PMC
August 2007

Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves.

Croat Med J 2007 Aug;48(4):478-85

International Commission on Missing Persons, Sarajevo, Bosnia and Herzegovina.

Aim: To quantitatively compare a silica extraction method with a commonly used phenol/chloroform extraction method for DNA analysis of specimens exhumed from mass graves.

Methods: DNA was extracted from twenty randomly chosen femur samples, using the International Commission on Missing Persons (ICMP) silica method, based on Qiagen Blood Maxi Kit, and compared with the DNA extracted by the standard phenol/chloroform-based method. The efficacy of extraction methods was compared by real time polymerase chain reaction (PCR) to measure DNA quantity and the presence of inhibitors and by amplification with the PowerPlex 16 (PP16) multiplex nuclear short tandem repeat (STR) kit.

Results: DNA quantification results showed that the silica-based method extracted on average 1.94 ng of DNA per gram of bone (range 0.25-9.58 ng/g), compared with only 0.68 ng/g by the organic method extracted (range 0.0016-4.4880 ng/g). Inhibition tests showed that there were on average significantly lower levels of PCR inhibitors in DNA isolated by the organic method. When amplified with PP16, all samples extracted by silica-based method produced 16 full loci profiles, while only 75% of the DNA extracts obtained by organic technique amplified 16 loci profiles.

Conclusions: The silica-based extraction method showed better results in nuclear STR typing from degraded bone samples than a commonly used phenol/chloroform method.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2080566PMC
August 2007

DNA identification of "Earthquake McGoon" 50 years postmortem.

J Forensic Sci 2007 Sep 23;52(5):1115-8. Epub 2007 Jul 23.

Armed Forces DNA Identification Laboratory, Armed Forces Institute of Pathology, AFIP Annex, 1413 Research Blvd., Rockville, MD 20850, USA.

This report describes the genetic identification of James "Earthquake McGoon" McGovern, a WWII fighter ace who perished in Laos while providing supplies to French troops during the French Indochina war. Because reference samples were unavailable for all of the potential casualties, testing of the entire mitochondrial genome, autosomal STRs and Y-chromosomal STRs was performed to increase the genetic information available for analysis. Kinship analyses performed on the evidentiary data and numerous indirect family references for McGovern excluded other possible casualties and definitively established McGovern's identity. This particular case demonstrates the practical utility of novel research technologies and aggressive genetic typing protocols in the identification of aged, degraded remains.
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http://dx.doi.org/10.1111/j.1556-4029.2007.00506.xDOI Listing
September 2007

High efficiency DNA extraction from bone by total demineralization.

Forensic Sci Int Genet 2007 Jun 12;1(2):191-5. Epub 2007 Mar 12.

Armed Forces DNA Identification Laboratory, 1413 Research Blvd., Bldg. 101, Rockville, MD 20850, United States.

In historical cases, missing persons' identification, mass disasters, and ancient DNA investigations, bone and teeth samples are often the only, and almost always the best, biological material available for DNA typing. This is because of the physical and chemical barrier that the protein:mineral matrix of bone poses to environmental deterioration and biological attack. Most bone extraction protocols utilized in the forensic community involve an incubation period of bone powder in extraction buffer for proteinase digestion, followed by the collection of the supernatant, and the disposal of large quantities of undissolved bone powder. Here we present an extremely efficient protocol for recovery of DNA by complete demineralization, resulting in full physical dissolution of the bone sample. This is performed in a manner that retains and concentrates all the reagent volume, for complete DNA recovery. For our study, we selected 14 challenging bone samples. The bones were extracted side-by-side with our new demineralization protocol and the standard extraction protocol in use at AFDIL. A real-time quantification assay based on the amplification of a 143 bp mtDNA fragment showed that this new demineralization protocol significantly enhances the quantity of DNA that can be extracted and amplified from degraded skeletal remains. We have used this technique to successfully recover authentic DNA sequences from extremely challenging samples that failed repeatedly using the standard protocol.
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http://dx.doi.org/10.1016/j.fsigen.2007.02.006DOI Listing
June 2007

Application of novel "mini-amplicon" STR multiplexes to high volume casework on degraded skeletal remains.

Forensic Sci Int Genet 2007 Jun 13;1(2):175-9. Epub 2007 Mar 13.

International Commission on Missing Persons, 45A Alipasina, 71000 Sarajevo, Bosnia and Herzegovina.

The International Commission on Missing Persons (ICMP) conducts high throughput STR profiling on degraded skeletal remains, primarily recovered from mass graves relating to conflicts from 1992 to 1999 in the former Yugoslavia. To date, over 11,000 individuals have been identified through comparison of bone profiles to a large database of profiles from family members of the missing. To increase success rates in STR recovery, three short amplicon STR multiplexes (a 7-plex, a 6-plex, and a 5-plex) have been devised and implemented. These target loci from large commercial multiplexes, with an average decrease in amplicon size of 144 bp. The ICMP "miniplexes" have proven to provide substantially greater recovery of DNA data from a certain subset of difficult samples. However, the circumstances under which miniplexes provide additional data are restricted, and their advantages do not outweigh those of large commercial multiplexes for a majority of cases. The miniplexes, however, also have a very powerful use in DNA testing to support large scale reassociation of commingled, partial skeletons recovered from secondary mass graves.
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http://dx.doi.org/10.1016/j.fsigen.2007.02.003DOI Listing
June 2007

Mitochondrial control region sequence variations in the Hungarian population: analysis of population samples from Hungary and from Transylvania (Romania).

Forensic Sci Int Genet 2007 Jun 20;1(2):158-62. Epub 2007 Apr 20.

Department of Haemogenetics, Institute for Forensic Sciences, Budapest, Hungary.

To assess the mitochondrial DNA polymorphisms of the Hungarian population in the Carpathian basin and to facilitate forensic mtDNA testing a collection of control region sequences were generated from two population samples from Hungary and from two Hungarian speaking populations from Transylvania (Romania). Entire control region sequencing was performed by an automated laboratory process and data export without any manual transcription. The random match probability and pairwise comparisons within and between the datasets is reported. This study highlights the importance of considering population structure when generating reference databases for forensic testing. Comparisons between our population samples indicate the need for heightened caution when sampling, and using mtDNA databases of small endogamous populations. The population data will be incorporated in the EMPOP database (www.empop.org).
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http://dx.doi.org/10.1016/j.fsigen.2007.03.001DOI Listing
June 2007

Development and expansion of high-quality control region databases to improve forensic mtDNA evidence interpretation.

Forensic Sci Int Genet 2007 Jun 13;1(2):154-7. Epub 2007 Mar 13.

Armed Forces DNA Identification Laboratory (AFDIL), 1413 Research Blvd., Rockville, MD 20850, USA.

In an effort to increase the quantity, breadth and availability of mtDNA databases suitable for forensic comparisons, we have developed a high-throughput process to generate approximately 5000 control region sequences per year from regional US populations, global populations from which the current US population is derived and global populations currently under-represented in available forensic databases. The system utilizes robotic instrumentation for all laboratory steps from pre-extraction through sequence detection, and a rigorous eight-step, multi-laboratory data review process with entirely electronic data transfer. Over the past 3 years, nearly 10,000 control region sequences have been generated using this approach. These data are being made publicly available and should further address the need for consistent, high-quality mtDNA databases for forensic testing.
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http://dx.doi.org/10.1016/j.fsigen.2007.01.019DOI Listing
June 2007