Solving abandoned cold cases with molecular testing

With its founding charter dating to 1632, Maryland is one of the earliest colonies in the US. Lost settlements and cemeteries have been hiding underground for centuries. So when a road is mapped through the landscape or backhoes break ground on new housing development, human remains can appear. It happens regularly. Most of the remains are partial – a skull, few teeth or a long bone. In one case the team recently worked on, natural erosion caused bones to begin leaching through a riverbank.

Consider these historical cases. The mystery of these remains is often greater than that of modern remains because researchers often have to reconstruct the 'person's identity from their bones and location alone.

At Towson University Human Remains Identification Laboratory (THRIL), forensic scientist Kelly Elkins is training undergraduate and graduate students to apply next-generation sequencing (NGS) to historical forensic samples that are difficult to conclude. DNA degrades over time, so extracting and amplifying genetic material requires the right skills and the right technology. The methods are also applicable to modern crime scenes where the DNA remnants are scant.

Elkins, a professor of chemistry at TU, believes it's essential to train the next generation of forensic scientists on the latest technologies. For a forensic scientist, having the skills to wield the best tools available will increase their chances of revealing the victim's details and perhaps bring closure to their loved ones.

Solving the mystery of a bone

In recent years, many traditional forensic methods have been discredited, which makes NGS especially important to modern labs like THRIL.

In 2009, a report by the National Academy of Sciences noted the lack of "peer-reviewed, published studies establishing the scientific bases and validity of many forensic methods,” including bite mark analysis, microscopic hair analysis, shoe print comparisons, handwriting comparisons, fingerprint examinations,firearms and tool mark examinations. “DNA was the only one left standing," Elkins says.

Founded in 2018, the THRIL lab is home to hands-on classes in advanced DNA sequencing methods, including Sanger sequencing, pyrosequencing, and massively parallel sequencing (MPS).

“DNA technology has been used in forensic settings for more than 20 years, but NGS expanded the number of loci from 24 to 231,” Elkins says. "We're able to extract DNA from human bone and tooth samples and determine the genotype as well as sex and ancestry. From the phenotype we can determine external features like eye color, skin color and hair color." They can even differentiate between identical twins.

The right equipment

"One of the challenges is obtaining high-enough quality DNA to obtain a full profile with these samples," she says. DNA extracted from bone and teeth is typically low quantity, and it's often low quality in historical contexts. Acidic, very wet or dry soil is also destructive and not ideal for sample recovery.

Similarly, quantity and quality factors are relevant to the forensics of modern sites as well, Elkins says. “Sometimes when DNA is old or improperly stored or just low in quantity from crime scenes, you don't get a complete profile using the traditional methods. NGS is much more sensitive. We can get more data out of degraded samples than ever before."

"We use a lot of QIAGEN equipment," Elkins says, “such as the BioRobot® EZ1 instrument for DNA extraction of six samples at a time. For our level of throughput, the instrument is absolutely perfect. Students get experience with robots, but we don't waste a lot of supplies - we only use one cartridge for one sample at a time."

They use one of their two QIAGEN Rotor-Gene Qs to amplify and quantify the DNA. "It is such a robust and reliable instrument," she says. "We primarily use them for commercial qPCR kits and for in-house assays that we've designed ourselves."

Also important are the Rotor-Gene ScreenClust HRM Software, a principal component analysis tool used for molecular genotype analyses, and the PyroMark sequencer for SNP genotyping and body fluid analysis.

There's also the MiSeq FGx, an MPS instrument tailored for forensic genomics. "In about 27 to 30 hours, we can sequence and analyze up to 96 samples with that instrument." That is important because student research projects can involve a high number of samples that need to be run in multiple replicates. "It's great to be able to put all of the class samples in the instrument at one time and run them over a weekend," she says. "They can analyze everything when they're back in class on Monday."

Exciting discoveries

"In some of the recent cases with our anthropologists, we have been able to use DNA to determine that we have African-American and European origin ancestors from the same site and we've had children as well as adults," Elkins says.

"We determined that several of those bones are from different individuals, but some of those bones were from the same individuals—the DNA profiles matched. We were able to determine how many individuals were at a site and compare that to the anthropologist's estimate. And so that was really exciting."

And there are some modern cold cases too. In one case, the forensic scientists analyzed the sample of spit out chewing gum that was collected in the woods near artifacts thought to be connected to a cold case. They were able to extract DNA from the gum, amplify it, and using the mitochondrial DNA analysis with some other assays, determine the individual’s haplotype and European ancestry.

While solving modern cold cases can bring closure to grieving families, closing historical cases can connect descendants to their ancestors and change our understanding of the past. "Not everyone's history was recorded," Elkins points out. "With the new technology, we're able to provide not only the ancestry but a bit of history. The way things were written may be different from the way they really were. I think we can add a lot to our understanding of colonial America."