Improved DNA Extraction and Illumina Sequencing of DNA Recovered from Aged Rootless Hair Shafts Found in Relics Associated with the Romanov Family.

This study describes an optimized DNA extraction protocol targeting ultrashort DNA molecules from single rootless hairs. It was applied to the oldest samples available to us: locks of hairs that were found in relics associated with the Romanov family. Published mitochondrial DNA genome sequences of Tsar Nicholas II and his wife, Tsarina Alexandra, made these samples ideal to assess this DNA extraction protocol and evaluate the types of genetic information that can be recovered by sequencing ultrashort fragments. Using this method, the mtGenome of the Tsarina's lineage was identified in hairs that were concealed in a pendant made by Karl Fabergé for Alexandra Feodorovna Romanov. In addition, to determine if the lock originated from more than one individual, two hairs from the locket were extracted independently and converted into Illumina libraries for shotgun sequencing on a NextSeq 500 platform. From these data, autosomal SNPs were analyzed to assess relatedness. The results indicated that the two hairs came from a single individual. Genetic testing of hairs that were found in the second artifact, a framed photograph of Louise of Hesse-Kassel, Queen of Denmark and maternal grandmother of Tsar Nicholas II, revealed that the hair belonged to a woman who shared Tsar Nicholas' maternal lineage, including the well-known point heteroplasmy at position 16169.

Mitochondrial DNA analysis of the putative skeletal remains of Sieur de Marle: Genetic support for anthropological assessment of biogeographic ancestry.

In 1932, seven burials were discovered on a Texas plantation that was originally the site of a 17th-century Caddo Indian village. Of the seven excavated graves, one set of remains (an adult male) was notably buried in a manner inconsistent with traditional Caddoan burial practices and has long been purported to be the remains of Sieur de Marle (a member of the French explorer La Salle's last expedition). Diary accounts of La Salle's expedition scribe report that Sieur de Marle died along a river near an Indian village during a trek to Canada to find help for colonists left behind at the ill-fated Fort St. Louis. Additionally, two lead projectiles recovered from the grave were ballistically analyzed and determined to be consistent with ammunition used in 17th-century weaponry. In the 1980s, anthropologists requested access to the remains for study, but the skull was missing. Cranial measurements recorded in 1940 and 1962 (by two independent anthropologists) were used to investigate the ancestry of this individual; and the Giles-Elliot (G-E) discriminant function was calculated to be 18.1, within the Anglo-European range. Dietary isotope testing on non-cranial skeletal elements determined that this unknown male's diet was rich in animal/marine protein sources, which differs appreciably from Caddo Indian populations of that time period. In order to genetically assess this individual's biogeographic ancestry and to provide further support that this individual is of European descent, mitochondrial DNA (mtDNA) sequencing was performed using the Applied Biosystems™ Precision ID mtDNA Whole Genome Panel. mtDNA sequencing of multiple sections from two different long bones yielded compiled results consistent with either Haplogroup H or R, both predominantly European mtDNA haplogroups. Further anthropological calculations were conducted using cranial measurements, FORDISC™ software, and discriminant function analysis. Two-way, four-way, and multigroup discriminant function analyses further classify this set of unidentified remains as being White (European) in origin, with posterior probabilities of 0.999, 0.881 and 0.986, respectively. Combined with historical records of Sieur de Marle's death, as well as overlays of historical and contemporary maps which demonstrate that the plantation site aligns with Joutel's diary accounts of de Marle's burial, these collective results support that these remains are of a European male and may possibly belong to this prominent member of La Salle's expedition team.

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory.

As a first step towards integrating next generation sequencing (NGS) technology into the FBI Laboratory's operational casework, the PowerSeq™ CRM Nested System, an NGS-based mitochondrial DNA (mtDNA) control region assay, was developmentally and internally validated. The validation studies were conducted in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) Validation Guidelines for Forensic DNA Analysis Methods, and the FBI's Quality Assurance Standards (QAS) for Forensic DNA Testing Laboratories. The assay was shown to be highly reproducible, with variant frequencies across intra and inter-run replicates of the same sample differing, on average, by just 0.3% for substitutions and point heteroplasmies and 1.5% for insertions and deletions. The assay was also shown to be extremely sensitive, yielding complete control region sequence data from as few as 2000 copies of mtDNA. This is a more than 20-fold increase in sensitivity when compared to the FBI Laboratory's current Sanger sequencing-based protocols and, based on mtDNA quantitation values of samples routinely encountered in mtDNA casework, suggests that the percentage of questioned samples from which full control region data can be recovered will increase from our current 20% to approximately 90% success with NGS technology. In addition, the assay requires on average only 30% of the extract volume typically required to develop control region profiles from degraded samples via Sanger sequencing. Overall, these studies establish the reliability of the PowerSeq™ CRM Nested System for accurate mtDNA control region typing and can serve as a model for laboratories seeking to validate NGS protocols for forensic mtDNA analysis.

Fragmented Nuclear DNA is the Predominant Genetic Material in Human Hair Shafts.

While shed hairs are one of the most commonly encountered evidence types, they are among the most limited in terms of DNA quantity and quality. As a result, nuclear DNA short tandem repeat (STR) profiling is generally unsuccessful and DNA testing of shed hair is instead performed by targeting the mitochondrial DNA control region. Although the high copy number of mitochondrial DNA relative to nuclear DNA routinely permits the recovery of mitochondrial DNA (mtDNA) data in these cases, mtDNA profiles do not offer the discriminatory power of nuclear DNA profiles. In order to better understand the total content and degradation state of DNA in single shed hairs and assess the feasibility of recovering highly discriminatory nuclear DNA data from this common evidence type, high throughput shotgun sequencing was performed on both recently collected and aged (approximately 50-year-old) hair samples. The data reflect trends that have been demonstrated previously with other technologies, namely that mtDNA quantity and quality decrease along the length of the hair shaft. In addition, the shotgun data reveal that nuclear DNA is present in shed hair and surprisingly abundant relative to mitochondrial DNA, even in the most distal fragments. Nuclear DNA comprised, at minimum, 88% of the total human reads in any given sample, and generally more than 95%. Here, we characterize both the nuclear and mitochondrial DNA content of shed hairs and discuss the implications of these data for forensic investigations.

Potential effects of ionizing radiation on the evidentiary value of DNA, latent fingerprints, hair, and fibers: A comprehensive review and new results.

An extensive literature review and new post-irradiation experimental results are presented of genotyping blood stains and hair, and physical examinations of latent fingerprints, hairs, and fibers. Results indicate that successful development of nuclear short tandem repeat (STR) and mitochondrial DNA sequence profiles from human blood and hair evidence is possible-up to a point-following exposure to gamma, neutron, beta, and alpha radiation at several levels that would most likely be present at this type of crime scene (i.e., a "dirty bomb," etc.). Commencing at gamma radiation levels between 90 and 900kGy, DNA analysis using conventional DNA techniques was unsuccessful. In general, irradiation negatively affected the quality of latent fingerprints. All four radiation types degraded most fingerprint samples at all doses; nevertheless, many fingerprints remained of value for potential use in comparison. Although variable from one hair to another, microscopic changes observed for all types and levels of irradiation could potentially result in false exclusions. Negligible microscopic changes were observed in papers and fibers (used as substrates for fingerprints and bloodstains) up to 90kGy gamma, but fluorescence of fibers began to change above that dose. Paper and fibers, as well as plastic evidence enclosures, became extremely brittle leading to breakage after a gamma dose of 900kGy.

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. Using the same amplicon product, replicate library sets were generated and sequenced at different laboratories, and analysis was performed in replicate using the CLC Genomics Workbench. Both sequencing sets resulted in 99.998% of positions with greater than 10X coverage when 96 samples (including controls) were multiplexed. Overall, 99.9996% concordance was observed between the NGS data and the STS data for the full mtGenome. The only "discordant" calls involved low level point heteroplasmies, with the differences resulting from stochastic variation and/or the increased sensitivity of NGS. Higher sensitivity also allowed for the detection of a mixed sample previously not detected with STS. Additionally, variant calls were reproducible between sequencing sets and between software analysis versions with the variant frequency only differing by 0.23% and 0.01%, respectively. Further validation studies and specialized software functionality tailored to forensic practice should facilitate the incorporation of NGS processing into standard casework applications. The data herein comprise the largest, and likely most thoroughly examined, complete mtGenome STS-NGS concordance dataset available.

Massively parallel sequencing of complete mitochondrial genomes from hair shaft samples.

Though shed hairs are one of the most commonly encountered evidence types, they are among the most limited in terms of DNA quantity and quality. As a result, DNA testing has historically focused on the recovery of just about 600 base pairs of the mitochondrial DNA control region. Here, we describe our success in recovering complete mitochondrial genome (mtGenome) data (∼16,569bp) from single shed hairs. By employing massively parallel sequencing (MPS), we demonstrate that particular hair samples yield DNA sufficient in quantity and quality to produce 2-3kb mtGenome amplicons and that entire mtGenome data can be recovered from hair extracts even without PCR enrichment. Most importantly, we describe a small amplicon multiplex assay comprised of sixty-two primer sets that can be routinely applied to the compromised hair samples typically encountered in forensic casework. In all samples tested here, the MPS data recovered using any one of the three methods were consistent with the control Sanger sequence data developed from high quality known specimens. Given the recently demonstrated value of complete mtGenome data in terms of discrimination power among randomly sampled individuals, the possibility of recovering mtGenome data from the most compromised and limited evidentiary material is likely to vastly increase the utility of mtDNA testing for hair evidence.