Identifying 140 Year-Old Remains Using Massively Parallel DNA Sequencing

The goal of forensic DNA testing of human skeletal remains is identification of the unknown individual. A variety of genetic markers can be used to achieve identification, including highly polymorphic autosomal short tandem repeat (STR) loci and lineage markers (Y-STRs and mtDNA). However, reference samples must be available for comparison for these markers to be informative. In mass disasters, missing persons cases, or cases involving historical/archaeological remains, sometimes there are no clues as to the person’s potential identity and/or there are no associations made with a reference sample or reference pedigree via a database search. In such scenarios, identification can be difficult or impossible using solely autosomal STRs and lineage markers. However, there are other genetic markers that can extend human identification capabilities, such as analysis of ancestry-informative markers and phenotype-informative single nucleotide polymorphisms (SNPs).

Written by: Angie Ambers, University of North Texas Health Science Center

Massively parallel sequencing (MPS) of ancestry- and phenotype-informative SNPs, with its expanded capacity for marker typing, offers the ability to develop investigative leads in such cases. Thus, more genetic information can be gleaned from a sample without further consumption of often very limited quantity and quality samples.

I have been involved in skeletal remains work for many years. I am fascinated with bones and their ability to survive for such long periods of time in harsh conditions, and I have long been a history buff (so I eagerly accept the challenge of the “few-and-far-between” historical cases when they come along). There is so much publicity tied to current/modern cases (understandably due to the emotional trauma and impact on victims and their families). However, it’s important to note that a primary goal of forensics is to answer questions and solve cases. Sometimes historical cases get overlooked because they fall into the category of being “non-probative” — i.e., all individuals involved may have long been deceased and are therefore not available to participate in court proceedings. However, as practitioners and fellow human beings we should ask the question — does the mere passage of time negate the value of a person’s life or lessen the impact of solving a mystery? In my opinion, these are the coldest of cold cases and still deserve our attention.

Characterization of Unidentified 140 Year-Old Human Skeletal Remains using Massively Parallel DNA Sequencing

In this study, MPS was used in an effort to help characterize 140-year-old human skeletal remains that were buried in an unmarked grave in Deadwood, South Dakota USA, a famous town of the American Old West.

In 1874, the discovery of gold in the Black Hills of South Dakota set off one of the last great gold rushes in America. In 1876, miners moved to the area and formally established the city of Deadwood, now a U.S. historical landmark. Deadwood’s original cemetery, Ingleside, was located near the town’s core business district and contained approximately 100 burials (although cemetery records are incomplete and some were buried in unmarked graves). In 1878, the individuals interred there were relocated to the hills above Deadwood and Mount Moriah Cemetery was established.

In 2012, a set of unidentified human skeletal remains were unearthed by a construction crew in Deadwood’s Presidential District, the original site of Ingleside Cemetery. South Dakota State archaeologists and historic preservation officials for the City of Deadwood recovered the skeleton from the site (with the exception of one tooth and a few bones from the hands and feet). Anthropological analyses indicated that the remains are consistent with a male of European ancestry (Caucasian) who was 18-24 years of age at the time of death and 65.7−70.7 inches tall.  No indications of the cause of death were evident in the skeletal samples. Forensic odontological analyses determined that this unknown individual was a habitual tobacco user and had nine dental fillings (3 gold, 6 tin/amalgam). The latter observation is indicative of some level of affluence/wealth, as most individuals in the late 19^th century would simply have had unhealthy teeth extracted.

In June 2014, the City of Deadwood and the Deadwood Historic Preservation Commission requested that the Institute of Applied Genetics (IAG) conduct DNA testing on the remains to provide confirmation of the European biogeographic ancestry suggested by anthropology and to identify phenotypic traits that might assist with identification. Given that the remains were in an unmarked grave and no investigative leads existed regarding his identity, Deadwood city officials were interested in the analysis of DNA markers that could help predict the individual’s ancestry and external physical traits.  Markers chosen for analysis included Y-STRs, Y-SNPs, ancestry-informative SNPs, phenotype-informative SNPs, and mitochondrial DNA (mtDNA).

This collaborative project was funded by the Institute of Applied Genetics (IAG) and the City of Deadwood Office of Historic Preservation. The skeletal remains were loaned to the IAG by the South Dakota State Historical Society-Archaeological Research Center for DNA testing. Deadwood City Archivist and historic preservation officer Michael Runge provided invaluable insights into the historical aspects of the case. South Dakota archaeologist and repository manager Katie Lamie provided information regarding burial excavation and handling of the remains prior to submission for DNA analyses.

The practices for minimizing contamination during the analysis of the Deadwood remains were the same contamination controls standardly recommended for archaeological and ancient DNA specimens. The right femur was provided to the IAG for DNA testing. Bone powder fractions from 4 sections of the femur were extracted for DNA and the following analyses were performed:

1. Y-STR typing with AmpFlSTR® Yfiler^™ and traditional CE
2. Massively parallel sequencing (MPS) with Illumina’s ForenSeq™ DNA Signature Prep Kit and MiSeq® Desktop Sequencer
3. Massively parallel sequencing (MPS) with ThermoFisher’s SNP panels [HID-Ion AmpliSeq^™ Identity Panel, HID-Ion AmpliSeq™ Ancestry Panel, and an External Variable Characteristics (EVC) prototype panel] and the Ion Torrent Personal Genome Machine® (PGM)
4. Mitochondrial DNA analysis of eleven regions with an in-house PCR multiplex assay

In an effort to learn more about the late-19^th-century human skeletal remains discovered at the site of Deadwood’s first cemetery, historic preservation officials enlisted a number of forensic specialists to conduct analyses on the remains that could assist in his identification. Since the individual was buried in an unmarked grave and no investigative leads existed regarding his identity, lineage testing and forensic DNA phenotyping (FDP) were performed to predict ancestry and external physical traits.

The Y-chromosome (Y-STR, Y-SNP) and mitochondrial DNA (mtDNA) profiles of the unidentified skeletal remains are consistent with the R1b and H1 haplogroups, respectively.  Both of these haplogroups are the most common ones in Western Europe. The ancestry-informative SNPs also indicated a European background. These genetic results are consistent with the findings of a previous anthropological report which determined that the Deadwood unidentified skeletal remains belong to a male of European ancestry (Caucasian). The phenotype-informative SNPs provided strong support that the individual had light red hair and brown eyes. This is the first known historical remains case that has been characterized with genetic panels designed specifically for forensic human identification purposes.

The results were highly informative. The study demonstrates the potential of MPS to analyze unidentified human skeletal remains and to provide substantially more genetic information from the same initial quantities of DNA sample as that of CE-based analyses. Using the Illumina^® ForenSeq™ DNA Signature Prep Kit, results were obtained for 25/26 Y-STRs, 88/95 human identity SNPs, 51/54 ancestry-informative SNPs, 24/24 phenotype-informative SNPs, 27/29 autosomal STRs (plus amelogenin), and 4/8 X-STRs. With the HID-Ion AmpliSeq^™ Identity Panel, results were obtained for 34/34 Y SNPs and 90/90 human identity SNPs. The HID-Ion AmpliSeq^™ Ancestry Panel yielded data for 164/165 ancestry-informative SNP markers assayed. Combined results for all MPS panels included genetic data for 25/26 Y-STRs, 34/34 Y SNPs, 165/165 ancestry-informative SNPs, 28/28 phenotype-informative SNPs, 102/102 human identity SNPs, 27/29 autosomal STRs (plus amelogenin), and 4/8 X-STRs (as well as eleven regions of mtDNA).

With its capacity for simultaneous analysis of a multitude of different types of DNA markers, MPS technology holds promise for use in the characterization of historical and archaeological remains, and in missing persons cases. In addition, in mass disasters or other types of cases where reference samples are not available/known, genetic markers such as ancestry-informative and phenotype-informative SNPs can provide data for craniofacial reconstructions that could be useful for positive identification.

What’s Next for You?

I am currently working on another interesting historical case involving several sets of skeletal remains associated with the last expedition of the French explorer La Salle. In 1684, French King Louis XIV sent La Salle across the ocean with four ships to find the mouth of the Mississippi River, establish a colony and trade routes, and locate Spanish silver mines. That plan was never realized. Instead, La Salle lost ships to pirates and disaster, sailed past his destination, and was murdered by his own men. In 1686, La Belle, the one remaining expedition ship, wrecked in a storm and sank to the muddy bottom of Matagorda Bay off the coast of Texas, where it rested undisturbed for over 300 years. In 1996, archaeologists located the 17th century ship and began a decades-long process of excavating the ship and recovering its contents. I recently received a grant from the Texas Historical Commission to perform DNA analyses on human skeletal remains recovered from the shipwreck and Fort St. Louis, the French colony established by the shipwreck survivors (who were shortly thereafter massacred by local Indians). Based on diaries, historical records, and collaborations with French historians, archaeologists and anthropologists from the Texas Historical Commission have ideas about the identities of the deceased individuals; the goal of the DNA testing is to provide positive identification and link the skeletal remains to living descendants.

The following are links to press releases and publicity surrounding the Deadwood case:

• Skeletal Remains Headed for DNA Testing, Black Hills Pioneer, April 2014
• Skeletal Remains Headed for DNA Testing, Forensic Magazine, April 2014
• Putting a Face on the Past: Modern Scientists Unraveling a Historic Mystery, Rapid City Journal, June 2014
• Scientists Unraveling a Historic Deadwood Mystery, Washington Times, July 2014
• Forensic DNA Testing of Human Remains Reveals More Clues, Black Hills Pioneer, January 2015

Dr. Angie Ambers received her Ph.D. in molecular biology from the University of North Texas (UNT) with an emphasis in forensic genetics and human identification. Her dissertation involved an investigation of methods (e.g., whole genome amplification, DNA repair) for improving autosomal and Y-STR typing of degraded and low copy DNA from human skeletal remains and environmentally-damaged biological materials. Dr. Ambers also has master’s degrees in forensic genetics from the University of North Texas Health Science Center and in criminology from the University of Texas at Arlington. Her thesis research involved developing and optimizing a DNA-based multiplex screening tool for the separation of fragmented and commingled skeletal remains. Since 2005, Dr. Ambers has been an adjunct professor at the University of North Texas (teaching molecular biology, genetics, heredity, and human anatomy and physiology). In 2008, she developed the curriculum for a course in forensic molecular biology, in which she teaches DNA analysis/methodology to undergraduate students enrolled in the FEPAC-accredited forensic science certificate program. Before pursuing her doctorate, Dr. Ambers was lead DNA analyst and lab manager of UNT’s DNA Sequencing Core Facility. Her latest work has involved DNA testing of various historical human skeletal remains, including those of an American Civil War guerrilla scout, several Finnish World War II soldiers, unidentified late-19^th century skeletal remains discovered in Deadwood, South Dakota, and unidentified skeletal remains from the 1974 Turkish invasion of Cyprus. Dr. Ambers also recently collaborated with the National Institute of Justice (NIJ) and the Forensic Technology Center of Excellence (FTCoE) to develop and disseminate a formal report on the use of familial DNA searching in casework. She is currently a postdoctoral fellow at the IAG, specializing in characterization and identification of historical and archaeological human skeletal remains.

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