Review of SNP assays for disaster victim identification: Cost, time, and performance information for decision-makers.

In mass disaster events, forensic DNA laboratories may be called upon to quickly pivot their operations toward identifying bodies and reuniting remains with family members. Ideally, laboratories have considered this possibility in advance and have a plan in place. Compared with traditional short tandem repeat (STR) typing, single nucleotide polymorphisms (SNPs) may be better suited to these disaster victim identification (DVI) scenarios due to their small genomic target size, resulting in an improved success rate in degraded DNA samples.

Unearthing who and Y at Harewood Cemetery and inference of George Washington's Y-chromosomal haplotype.

An excavation conducted at Harewood Cemetery to identify the unmarked grave of Samuel Washington resulted in the discovery of burials presumably belonging to George Washington's paternal grandnephews and their mother, Lucy Payne. To confirm their identities this study examined Y-chromosomal, mitochondrial, and autosomal DNA from the burials and a living Washington descendant. The burial's Y-STR profile was compared to FamilyTreeDNA's database, which resulted in a one-step difference from the living descendant and an exact match to another Washington.

Complete Mitochondrial DNA Genome Variation in the Swedish Population.

The development of complete mitochondrial genome (mitogenome) reference data for inclusion in publicly available population databases is currently underway, and the generation of more high-quality mitogenomes will only enhance the statistical power of this forensically useful locus. To characterize mitogenome variation in Sweden, the mitochondrial DNA (mtDNA) reads from the SweGen whole genome sequencing (WGS) dataset were analyzed. To overcome the interference from low-frequency nuclear mtDNA segments (NUMTs), a 10% variant frequency threshold was applied for the analysis.

Evaluating the Usefulness of Human DNA Quantification to Predict DNA Profiling Success of Historical Bone Samples.

This study assessed the usefulness of DNA quantification to predict the success of historical samples when analyzing SNPs, mtDNA, and STR targets. Thirty burials from six historical contexts were utilized, ranging in age from 80 to 800 years postmortem. Samples underwent library preparation and hybridization capture with two bait panels (FORCE and mitogenome), and STR typing (autosomal STR and Y-STR).

The Value of Whole-Genome Sequencing for Mitochondrial DNA Population Studies: Strategies and Criteria for Extracting High-Quality Mitogenome Haplotypes.

Whole-genome sequencing (WGS) data present a readily available resource for mitochondrial genome (mitogenome) haplotypes that can be utilized for genetics research including population studies. However, the reconstruction of the mitogenome is complicated by nuclear mitochondrial DNA (mtDNA) segments (NUMTs) that co-align with the mtDNA sequences and mimic authentic heteroplasmy. Two minimum variant detection thresholds, 5% and 10%, were assessed for the ability to produce authentic mitogenome haplotypes from a previously generated WGS dataset.

Ancient DNA Methods Improve Forensic DNA Profiling of Korean War and World War II Unknowns.

The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old.

The FORCE Panel: An All-in-One SNP Marker Set for Confirming Investigative Genetic Genealogy Leads and for General Forensic Applications.

The FORensic Capture Enrichment (FORCE) panel is an all-in-one SNP panel for forensic applications. This panel of 5422 markers encompasses common, forensically relevant SNPs (identity, ancestry, phenotype, X- and Y-chromosomal SNPs), a novel set of 3931 autosomal SNPs for extended kinship analysis, and no clinically relevant/disease markers. The FORCE panel was developed as a custom hybridization capture assay utilizing ~20,000 baits to target the selected SNPs.

Extended kinship analysis of historical remains using SNP capture.

DNA-assisted identification of historical remains requires the genetic analysis of highly degraded DNA, along with a comparison to DNA from known relatives. This can be achieved by targeting single nucleotide polymorphisms (SNPs) using a hybridization capture and next-generation sequencing approach suitable for degraded skeletal samples. In the present study, two SNP capture panels were designed to target ~ 25,000 (25 K) and ~ 95,000 (95 K) nuclear SNPs, respectively, to enable distant kinship estimation (up to 4th degree relatives).

Capture enrichment and massively parallel sequencing for human identification.

In the past decade, hybridization capture has gained attention within the forensic field for its possible use in human identification. One of the primary benefits to capture enrichment is its applicability to degraded DNA fragments that, due to their reduced size, are not amenable to traditional PCR enrichment techniques. Hybridization capture is typically introduced after genomic library preparation of extracted DNA templates for the subsequent enrichment of mitochondrial DNA or single nucleotide polymorphisms within the nuclear genome.

Platinum-Quality Mitogenome Haplotypes from United States Populations.

A total of 1327 platinum-quality mitochondrial DNA haplotypes from United States (U.S.) populations were generated using a robust, semi-automated next-generation sequencing (NGS) workflow with rigorous quality control (QC).

Next generation sequencing of STR artifacts produced from historical bone samples.

STR artifacts are commonly observed in electrophoretic data and can complicate interpretation of the profiles produced. Even when a consensus approach is applied, reproducible artifacts have the potential to convolute the analysis. DNA obtained from historical bone samples is often heavily degraded and damaged, requiring the use of more sensitive procedures to increase allele recovery.

Pathogenic Variant Filtering for Mitochondrial Genome Haplotype Reporting.

Given the enhanced discriminatory power of the mitochondrial DNA (mtDNA) genome (mitogenome) over the commonly sequenced control region (CR) portion, the scientific merit of mitogenome sequencing is generally accepted. However, many laboratories remain beholden to CR sequencing due to privacy policies and legal requirements restricting the use of disease information or coding region (codR) information. In this report, we present an approach to obviate the reporting of sensitive codR data in forensic haplotypes.

A Forensic Genomics Approach for the Identification of Sister Marija Crucifiksa Kozulić.

Sister Marija Krucifiksa Kozulić (1852-1922) was a Croatian nun who is in consideration for beatification by the Vatican, which is facilitated by the identification of her 20th-century remains. Sister Marija was buried in a tomb in Rijeka, Croatia, along with other nuns including her biological sister, Tereza Kozulić (1861-1933). When the remains were exhumed in 2011, they were found in a deteriorated state and commingled with several other sets of remains.

Mitochondrial DNA haplogrouping to assist with the identification of unknown service members from the World War II Battle of Tarawa.

The World War II Battle of Tarawa, 1943, was a devastating conflict that resulted in losses of more than 1100 American and 4690 Japanese troops. The United States government aims to identify and repatriate the remains of all missing American service members through the Defense Prisoner of War/Missing in Action (POW/MIA) Accounting Agency (DPAA) and its partners such as the Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory (AFMES-AFDIL). Remains associated with the Battle of Tarawa have been recovered from field excavations conducted by History Flight, a DPAA strategic partner, as well as from the National Memorial Cemetery of the Pacific (NMCP) in Hawaii where unknowns have been disinterred for identification.

Impact of the sequencing method on the detection and interpretation of mitochondrial DNA length heteroplasmy.

Advancements in sequencing technologies allow for rapid and efficient analysis of mitochondrial DNA (mtDNA) in forensic laboratories, which is particularly beneficial for specimens with limited nuclear DNA. Next generation sequencing (NGS) offers higher throughput and sensitivity over traditional Sanger-type sequencing (STS) as well as the ability to quantitatively analyze the data. Changes in sample preparation, sequencing method and analysis required for NGS may alter the mtDNA haplotypes compared to previously generated STS data.

Resolving mitochondrial haplogroups B2 and B4 with next-generation mitogenome sequencing to distinguish Native American from Asian haplotypes.

Mitochondrial haplogroup information can be useful in forensic contexts that rely primarily on mitochondrial DNA (mtDNA) testing, which often involve limited or degraded DNA. Due to the phylogeographic patterning of mtDNA in human populations, mitochondrial haplogroups are indicative of maternal ancestry (as mtDNA is a maternally inherited marker). In certain circumstances, maternal ancestry inferred from mitochondrial haplogrouping could be beneficial to forensic investigations.

Mitochondrial DNA control region variation in Lebanon, Jordan, and Bahrain.

This study investigated the mitochondrial DNA (mtDNA) control region variation in Middle Eastern populations (610 individuals from Lebanon, Jordan and the Kingdom of Bahrain) for which population data are scarce. FST comparison among populations revealed that there are significant differences in mtDNA distributions between Bahrain and the two other populations, while Lebanon and Jordan showed no significant differences. This was also reflected by the distribution of the observed lineages that differed prominently between Bahrain and the other two investigated populations.

Bioinformatic removal of NUMT-associated variants in mitotiling next-generation sequencing data from whole blood samples.

Nuclear mitochondrial DNA segments (NUMTs) have arisen because of the transposition of segments of the mitochondrial DNA genome (mitogenome) into the nuclear genome. When using a "mitotiling" strategy, NUMTs may be more readily amplified when targeting the entire mitogenome compared to the control region, as hundreds of primers are required for complete sequencing coverage. In samples with a high percentage of nuclear DNA copies per cell, such as whole blood, NUMT coenrichment may be exacerbated.

Repair of DNA damage caused by cytosine deamination in mitochondrial DNA of forensic case samples.

DNA sequence damage from cytosine deamination is well documented in degraded samples, such as those from ancient and forensic contexts. This study examined the effect of a DNA repair treatment on mitochondrial DNA (mtDNA) from aged and degraded skeletal samples. DNA extracts from 21 non-probative, degraded skeletal samples (aged 50-70 years) were utilized for the analysis.

Developmental validation of a Nextera XT mitogenome Illumina MiSeq sequencing method for high-quality samples.

Generating mitochondrial genome (mitogenome) data from reference samples in a rapid and efficient manner is critical to harnessing the greater power of discrimination of the entire mitochondrial DNA (mtDNA) marker. The method of long-range target enrichment, Nextera XT library preparation, and Illumina sequencing on the MiSeq is a well-established technique for generating mitogenome data from high-quality samples. To this end, a validation was conducted for this mitogenome method processing up to 24 samples simultaneously along with analysis in the CLC Genomics Workbench and utilizing the AQME (AFDIL-QIAGEN mtDNA Expert) tool to generate forensic profiles.

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.

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.

A performance evaluation of Nextera XT and KAPA HyperPlus for rapid Illumina library preparation of long-range mitogenome amplicons.

Next-generation sequencing (NGS) facilitates the rapid and high-throughput generation of human mitochondrial genome (mitogenome) data to build population and reference databases for forensic comparisons. To this end, long-range amplification provides an effective method of target enrichment that is amenable to library preparation assays employing DNA fragmentation. This study compared the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and the KAPA HyperPlus Library Preparation Kit (Kapa Biosystems, Wilmington, MA) for enzymatic fragmentation and indexing of ∼8500bp mitogenome amplicons for Illumina sequencing.

Concordance and reproducibility of a next generation mtGenome sequencing method for high-quality samples using the Illumina MiSeq.

Sanger-type sequencing (STS) of mitochondrial DNA (mtDNA), specifically the control region (CR), is routinely employed in forensics in human identification and missing persons scenarios. Yet next-generation sequencing (NGS) has the potential to overcome some of the major limitations of STS processing, permitting reasonable paths forward for full mitochondrial genome (mtGenome) sequencing, while also offering higher-throughput and higher sensitivity capabilities. To establish the accuracy and reproducibility of NGS for the development of mtDNA data, 90 DNA extracts that were previously used to generate forensic quality full mtGenomes using STS were sequenced using Nextera XT library preparation and the Illumina MiSeq.

Full mtGenome reference data: development and characterization of 588 forensic-quality haplotypes representing three U.S. populations.

Though investigations into the use of massively parallel sequencing technologies for the generation of complete mitochondrial genome (mtGenome) profiles from difficult forensic specimens are well underway in multiple laboratories, the high quality population reference data necessary to support full mtGenome typing in the forensic context are lacking. To address this deficiency, we have developed 588 complete mtGenome haplotypes, spanning three U.S.