Armed Forces DNA Identification Laboratory Research Articles

Advancing mitochondrial genome data interpretation in missing persons casework

Massively parallel sequencing techniques have made possible the adoption of mitogenome sequencing for missing persons casework. In order to expand the family pedigree and population data necessary for sequence comparisons and statistical analysis, complete mitochondrial genome (mitogenome) haplotypes were generated by the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory using an automated laboratory workflow and robust analysis pipeline. Over 600 CEPH family pedigree samples from 87 lineages were processed in order to characterize the expected number of differences in mitogenome sequences between maternal relatives. Although stochastic artifacts in the CEPH cell lines elevated the point heteroplasmy rate three-fold compared to population sample data (1.8 vs. 0.46 point heteroplasmies per individual, on average), the CEPH family sequences exhibited a maximum of two substitutions between any two maternal relatives in nearly 500 generational events. The family study furthermore showed that elevating the variant detection threshold may create false substitutions at heteroplasmic sites. Results from an initial 2000 reference database samples indicate a first-pass success rate of 85%, with 97% unique mitogenome haplotypes. These data demonstrate advancements made toward the interpretation of mitogenome sequence data in missing persons casework.

Extraction of DNA from skeletal remains buried in acidic soils

The Armed Forces Medical Examiner System – Armed Forces DNA Identification Laboratory (AFMES-AFDIL) and the Defense POW/MIA Accounting Agency (DPAA) are partners in the identification of missing United States service members. Skeletal materials are recovered world-wide from past military conflicts; however, this project examines the relative success of skeletal materials recovered from Southeast Asia. The soils in Southeast Asia are known to be largely acidic, with the majority being classified as acrisol or gleysol (pH4.4–7.2). Acidic soils are thought to compromise the structural integrity of buried skeletal materials leading to a degradation of the DNA found within. This study evaluates whether this idea is apocryphal or if acidic soils truly have an impact.

Sequencing of full mitochondrial genomes for NIST population samples

The U.S. National Institute of Standards and Technology in collaboration with the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory has undertaken a population sequencing study of 607 mitochondrial genomes (mtGenomes). The goal of the study is to increase the availability of high-quality whole mtGenome sequences for haplotype frequency estimations, allowing the increased discrimination power of the entire extranuclear genome to be used. Whole mtGenome sequencing is enabled by next generation sequencing workflows, which are vastly simplified compared to Sanger-type sequencing. Here we evaluated the relative performance of mtGenome versus sequencing the control region of the mtGenome alone.

Complete Mitochondrial Genome Sequencing of a Burial from a Romano-Christian Cemetery in the Dakhleh Oasis, Egypt: Preliminary Indications.

The curse of ancient Egyptian DNA was lifted by a recent study which sequenced the mitochondrial genomes (mtGenome) of 90 ancient Egyptians from the archaeological site of Abusir el-Meleq. Surprisingly, these ancient inhabitants were more closely related to those from the Near East than to contemporary Egyptians. It has been accepted that the timeless highway of the Nile River seeded Egypt with African genetic influence, well before pre-Dynastic times. Here we report on the successful recovery and analysis of the complete mtGenome from a burial recovered from a remote Romano–Christian cemetery, Kellis 2 (K2). K2 serviced the ancient municipality of Kellis, a village located in the Dakhleh Oasis in the southwest desert in Egypt. The data were obtained by high throughput sequencing (HTS) performed independently at two ancient DNA facilities (Armed Forces DNA Identification Laboratory, Dover, DE, USA and Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA). These efforts produced concordant haplotypes representing a U1a1a haplogroup lineage. This result indicates that Near Eastern maternal influence previously identified at Abusir el-Meleq was also present further south, in ancient Kellis during the Romano–Christian period.

AQME: A forensic mitochondrial DNA analysis tool for next-generation sequencing data

The feasibility of generating mitochondrial DNA (mtDNA) data has expanded considerably with the advent of next-generation sequencing (NGS), specifically in the generation of entire mtDNA genome (mitogenome) sequences. However, the analysis of these data has emerged as the greatest challenge to implementation in forensics. To address this need, a custom toolkit for use in the CLC Genomics Workbench (QIAGEN, Hilden, Germany) was developed through a collaborative effort between the Armed Forces Medical Examiner System − Armed Forces DNA Identification Laboratory (AFMES-AFDIL) and QIAGEN Bioinformatics. The AFDIL-QIAGEN mtDNA Expert, or AQME, generates an editable mtDNA profile that employs forensic conventions and includes the interpretation range required for mtDNA data reporting. AQME also integrates an mtDNA haplogroup estimate into the analysis workflow, which provides the analyst with phylogenetic nomenclature guidance and a profile quality check without the use of an external tool. Supplemental AQME outputs such as nucleotide-per-position metrics, configurable export files, and an audit trail are produced to assist the analyst during review. AQME is applied to standard CLC outputs and thus can be incorporated into any mtDNA bioinformatics pipeline within CLC regardless of sample type, library preparation or NGS platform.An evaluation of AQME was performed to demonstrate its functionality and reliability for the analysis of mitogenome NGS data. The study analyzed Illumina mitogenome data from 21 samples (including associated controls) of varying quality and sample preparations with the AQME toolkit. A total of 211 tool edits were automatically applied to 130 of the 698 total variants reported in an effort to adhere to forensic nomenclature. Although additional manual edits were required for three samples, supplemental tools such as mtDNA haplogroup estimation assisted in identifying and guiding these necessary modifications to the AQME-generated profile. Along with profile generation, AQME reported accurate haplogroups for 18 of the 19 samples analyzed. The single errant haplogroup assignment, although phylogenetically close, identified a bug that only affects partial mitogenome data. Future adjustments to AQME’s haplogrouping tool will address this bug as well as enhance the overall scoring strategy to better refine and automate haplogroup assignments. As NGS enables broader use of the mtDNA locus in forensics, the availability of AQME and other forensic-focused mtDNA analysis tools will ease the transition and further support mitogenome analysis within routine casework. Toward this end, the AFMES-AFDIL has utilized the AQME toolbox in conjunction with the CLC Genomics Workbench to successfully validate and implement two NGS mitogenome methods.

Performance evaluation of a mitogenome capture and Illumina sequencing protocol using non-probative, case-type skeletal samples: Implications for the use of a positive control in a next-generation sequencing procedure

Next-generation ancient DNA technologies have the potential to assist in the analysis of degraded DNA extracted from forensic specimens. Mitochondrial genome (mitogenome) sequencing, specifically, may be of benefit to samples that fail to yield forensically relevant genetic information using conventional PCR-based techniques. This report summarizes the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory’s (AFMES-AFDIL) performance evaluation of a Next-Generation Sequencing protocol for degraded and chemically treated past accounting samples. The procedure involves hybridization capture for targeted enrichment of mitochondrial DNA, massively parallel sequencing using Illumina chemistry, and an automated bioinformatic pipeline for forensic mtDNA profile generation. A total of 22 non-probative samples and associated controls were processed in the present study, spanning a range of DNA quantity and quality. Data were generated from over 100 DNA libraries by ten DNA analysts over the course of five months.The results show that the mitogenome sequencing procedure is reliable and robust, sensitive to low template (one ng control DNA) as well as degraded DNA, and specific to the analysis of the human mitogenome. Haplotypes were overall concordant between NGS replicates and with previously generated Sanger control region data. Due to the inherent risk for contamination when working with low-template, degraded DNA, a contamination assessment was performed. The consumables were shown to be void of human DNA contaminants and suitable for forensic use. Reagent blanks and negative controls were analyzed to determine the background signal of the procedure. This background signal was then used to set analytical and reporting thresholds, which were designated at 4.0X (limit of detection) and 10.0X (limit of quantiation) average coverage across the mitogenome, respectively. Nearly all human samples exceeded the reporting threshold, although coverage was reduced in chemically treated samples resulting in a ∼58% passing rate for these poor-quality samples. A concordance assessment demonstrated the reliability of the NGS data when compared to known Sanger profiles. One case sample was shown to be mixed with a co-processed sample and two reagent blanks indicated the presence of DNA above the analytical threshold. This contamination was attributed to sequencing crosstalk from simultaneously sequenced high-quality samples to include the positive control. Overall this study demonstrated that hybridization capture and Illumina sequencing provide a viable method for mitogenome sequencing of degraded and chemically treated skeletal DNA samples, yet may require alternative measures of quality control.

Field Contamination of Skeletonized Human Remains with Exogenous DNA

The Armed Forces DNA Identification Laboratory reports the mitochondrial DNA (mtDNA) sequences of over 800 skeletal samples a year for the Joint POW/MIA Accounting Command-Central Identification Laboratory. These sequences are generated from degraded skeletal remains that are presumed to belong to U.S. service members missing from past military conflicts. In the laboratory, it is possible to control for contamination of remains; however, in the field, it can be difficult to prevent modern DNA from being transferred to skeletal elements and being carried forward through the analysis process. Four such cases are described here along with the controls in place in the laboratory to eliminate the possibility of the exogenous DNA being reported as authentic. In each case, the controls implemented by the laboratories prevented the false reporting of contaminant exogenous DNA from remains that were either faunal or human, but lacked endogenous DNA.

Comparison of DNA extraction methods for identification of human remains

After mass disasters, where the bodies of victims have been degraded, soft tissues are often completely lost, with the result that bones and teeth are all that remain for identification. The aim of this study was to investigate three DNA extraction methods that are currently used with degraded bones in forensic contexts: a silica-based extraction protocol used by the International Commission for Missing Persons (ICMP), a total demineralisation method used by the US Armed Forces DNA Identification Laboratory (AFDIL) and a standard organic DNA extraction method used by the Australian Federal Police (AFP). The methods were compared by real-time PCR quantitation and STR profiling. The silica and organic methods were not significantly different for either of these measures for bone but were both significantly more successful than the demineralisation method. Bovine serum albumin (BSA) was found to significantly improve the DNA yields from real-time PCR and the number of reportable alleles from STR typing, especially when using bone samples.

Arm in the Ice

In this article the author discusses how fingerprint- and DNA-identification techniques helped to solve a mystery from a 60-year-old airplane crash. She provides an overview of the March 12, 1948 crash of Northwest Airlines flight 4422 into Mount Sanford in the Wrangell Mountains in eastern Alaska that killed 24 passengers, including merchant marines returning to the U.S. from Shanghai, China and Northwest crew members. Topics include the discovery of an arm in 1999 by former U.S. Air Force pilots Kevin McGregor and Marc Millican and the development of a DNA-identification technique by researcher Odile Loreille of the Armed Forces DNA Identification Laboratory in Rockville, Maryland, that uses a demineralization process to dissolve bone matrix for mitochondrial DNA (mtDNA) analysis.

Estimation of DNA Concentration in Forensic Samples Prior to Multiplex PCR Amplification

INTRODUCTIONThis protocol describes the use of quantitative polymerase chain reaction (qPCR) for quantitation of DNA in forensic samples prior to PCR amplification. It is adapted from the Armed Forces DNA Identification Laboratory (Rockville, MD) Standard Operating Procedure.

Sampling of the cranium for mitochondrial DNA analysis of human skeletal remains

Sampling of cranial fragments for mitochondrial DNA (mtDNA) analysis is a common practice for identification of skeletonized human remains. The Armed Forces DNA Identification Laboratory (AFDIL) and Joint POW/MIA Accounting Command – Central Identification Laboratory (JPAC-CIL) work in concert to identify the remains of US service members killed in past military conflicts. When dealing with samples taken from remains that are commingled or lack a secure archaeological context, multiple elements must be sampled from the same burial or case. In such cases, the cranium is often fragmentary. Previous work examined the success rate of approximately 4000 skeletal elements for sequencing of mtDNA. The approximately 50% success of the crania seemed anomalous considering the frequency with which they are sampled. Subsequent studies of the 558 cranial fragments tested from 1992 to August 2009 were done to examine the independent rates of success of the portions of the skull. It was found that each yielded reportable mtDNA sequence at rates that exhibited statistically significant differences.

Diagnosing post-mortem treatments which inhibit DNA amplification from US MIAs buried at the Punchbowl

The US military is committed to recovering and identifying the remains of unknown military service members. Casualties of the Korean War were exhumed from the National Memorial Cemetery of the Pacific, or Punchbowl, and submitted to the Armed Forces DNA Identification Laboratory (AFDIL) for mtDNA sequencing. Contrary to AFDIL's experience on other samples from this era, most failed to yield amplifiable DNA. Suspicion fell on mortuary practices that may have been applied to the remains, evidenced by a white powder found with the bones, and general records suggesting the use of formaldehyde-based stablizing agents. To improve the chances of successful identification of the unknown individuals, we looked for the causes underlying this failure. We did this by examining the state of the collagen, the most abundant biomolecule in bone, by using differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). The DSC analyses showed collagens with a range of different thermal stabilities. When these results were compared with the DNA amplification results, a clear correlation between elevated thermal stability and amplification failure was evident. TEM analysis revealed that fibril integrity was maintained after thermal and acid treatments in the samples which failed amplification. Together these two approaches implicate a stabilization agent as the cause of problems with DNA analysis, presumably due to excessive cross-linking. Following the initial study, the ability of DSC to rapidly identify problem samples was tested in a blind study of 14 samples, the method successfully identifying all the problematic samples from Punchbowl. Within this unusual context, DSC analysis is a useful method to assess the likelihood of successful DNA extraction and amplification.

A Modified Mini-Primer Set for Analyzing Mitochondrial DNA Control Region Sequences from Highly Degraded Forensic Samples

To facilitate the analysis of mitochondrial DNA (mtDNA) control region sequences from highly degraded skeletal remains, a modified mini-primer set was designed to overcome the limitations of the Armed Forces DNA Identification Laboratory (AFDIL) mini-primer set. This modified mini-primer set is less affected by nucleotide variability and PCR amplification conditions than the AFDIL mini-primer set, and was able to amplify the mtDNA sequences of 55-year-old skeletal remains with high efficiency, indicating that it is a useful tool for analyzing mtDNA control region sequences from highly degraded forensic samples.

Authors' Response

Journal of Forensic SciencesVolume 52, Issue 2 p. 494-494 Authors' Response John M. Butler Ph.D., John M. Butler Ph.D. National Institute of Standards and, Technology Biochemical Science Division, 100 Bureau Drive, M/S 8311, Gaithersburg, MD 20899-8311, E-mail: [email protected]Search for more papers by this authorMichael D. Coble Ph.D., Michael D. Coble Ph.D. Armed Forces DNA Identification Laboratory, Research Section, 1413 Research Boulevard, Building 101, Rockville, MD 20850Search for more papers by this author John M. Butler Ph.D., John M. Butler Ph.D. National Institute of Standards and, Technology Biochemical Science Division, 100 Bureau Drive, M/S 8311, Gaithersburg, MD 20899-8311, E-mail: [email protected]Search for more papers by this authorMichael D. Coble Ph.D., Michael D. Coble Ph.D. Armed Forces DNA Identification Laboratory, Research Section, 1413 Research Boulevard, Building 101, Rockville, MD 20850Search for more papers by this author First published: 16 February 2007 https://doi.org/10.1111/j.1556-4029.2007.00390.xCitations: 18Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume52, Issue2March 2007Pages 494-494 RelatedInformation

Forensics

Forensics