The advent of DNA-profiling changed forensic science forever. For the next 30 years, the basic standard of matching a profile with a suspect using analysis of STR amplicons by capillary electrophoresis (CE) remained constant. Recent developments in next-generation sequencing (NGS) have now transformed the landscape of DNA-profiling itself. NGS allows for the differentiation of STR alleles by sequence rather than size and this offers several important advantages over CE.
With NGS, smaller amplicons can be targeted, leading to improved analysis of mixed, degraded or limited samples. Access via NGS to sequence-level information also leads to increased allelic diversity. This in turn improves the power of discrimination of an STR profile that is obtained using NGS compared to CE. In addition to analyzing STRs, NGS can detect identity-specific SNPs, estimate externally visible characteristics, analyze mitochondrial DNA and provide both short- and long-range kinship information. All these attributes add tremendous value to forensic science.
Traditional kinship DNA analysis targets STRs. Improved NGS methods generate unique DNA profiles with SNPs to determine degrees of relatedness between individuals. A profile in a direct-to-consumer genetic genealogy DNA database such as GEDmatch can be combined with traditional genealogy methods to identify distant relatives and give names to unidentified human remains (UHR). Forensic investigative genetic genealogy (FIGG) generates leads in unsolved violent criminal cases. NGS workflows such as ForenSeq Kintelligence support FIGG with small amplicons that specifically target human DNA and interrogate only the parts of the genome that are relevant to kinship determination. Genetic associations out to third degree are possible.
Our conversation introduces Dr. Monika Stoljarova-Bibb who discovered forensic science through an internship in Texas and is now a researcher in the Estonian Forensic Science Institute. Dr. Stoljarova discusses the application of distant kinship in forensic genetics, including FIGG cases, and outlines the reasons why NGS was introduced into her lab. Dr. Stoljarova describes the valuable contribution of NGS in a collaboration with law enforcement to uncover an unidentified serial rapist and how NGS assisted police dealing with UHR recovered from the sea.
Tell us about your background and how you became interested in forensic science?
It all started with me receiving the Baltic-American Freedom Foundation scholarship for a year-long internship at a US institution. At that point, in the middle of my Master’s studies in Genetics, I was looking for a discipline to specialize in. My university supervisor advised me to contact Dr. Bruce Budowle at the Center for Human Identification, located within the University of North Texas Health Science Center. I didn’t know much about forensic genetics, but like most people, I thought the field sounded exciting. Who wouldn’t want to catch a bad guy from a long-lost hair found at a crime scene? After an interview with Dr. Budowle, a lot of paperwork, and reading some manuscripts on massively parallel sequencing (MPS) and forensics, I set off to Texas. It was one of the best decisions I made in my life.
After my internship at Dr. Budowle's lab, under the strict supervision of Dr. Jonathan King, I returned to TalTech (Tallinn University of Technology) and graduated with my MSc, and four years later, with my PhD in Natural Sciences. The focus of my research was the use of MPS in forensics. In 2022, Maarja Sadam, the Head of the DNA Department at the Estonian Forensic Science Institute, invited me to join the team as a research scientist.
Now, I am mainly involved in our current R&D projects. These focus on the use of distant kinship in forensic genetics, including forensic investigative genetic genealogy (FIGG). However, I am also going through DNA expert training and, therefore, doing real casework.
What made your lab decide to bring in NGS?
The number of markers that next-generation sequencing (NGS) allows you to type is much higher compared to capillary electrophoresis (CE). With these markers, you can estimate phenotype, biogeographic origin, distant kinship, and perform FIGG. Additionally, there is mitochondrial genome (mtGenome) analysis beyond the control regions, microbiome analysis, RNA analysis for tissue identification and age prediction. Not to mention, NGS often yields much better results with highly degraded DNA and complicated mixtures compared to traditional CE.
We wanted to increase our capabilities in the analysis of samples from complicated cases.
Monika’s colleague – Maile Kohtla, a Post-PCR lab specialist Please describe the types of cases your laboratory utilizes NGS for and what does NGS offer for these cases that traditional methods do not?
Cases with distant and complicated kinships are the ones that we use NGS the most. A lot of NGS power has gone into FIGG cases as well. We have used the phenotype and biogeographic origin estimations for unidentified human remains and crime scene samples with no suspects.
Which QIAGEN products do you use in your NGS workflow and what do you like about the products?
ForenSeq DNA Signature Prep Kit was the first NGS product that we tested. Now it is our validated and accredited method. We like the sensitivity and the relatively easy workflow.
We started working with ForenSeq Kintelligence Kit last summer. For now, we have analyzed samples from 14 unidentified human remains (UHR) cases. Using whole genome sequencing (WGS) for generating DNA profiles for FIGG is great, but for a midsize casework lab, like we are, it is rare to have a WGS platform. Finding a MiSeq FGx in a lab like ours is becoming more and more common. With Kintelligence (and MiSeq FGx) we are able to generate DNA profiles that can be used for FIGG, and all this data is produced in house. The workflow is relatively easy, including uploading of the profiles to GEDmatch Pro.
We like that it works well with degraded DNA. We got a 98% of Kintelligence SNPs for one bone sample that didn’t give much with microarray nor WGS analysis.
Can you tell us about your laboratory’s recent case success using NGS?
We have an active case involving a serial rapist who is still at large. The case is very complicated because the perpetrator is leaving almost no evidence for the police to work with, including DNA. For a long time, all the information that the police had was the descriptions of the perpetrator given by the victims. This, as is known, might be misleading. By using NGS, we were able to confirm the statements of the victims regarding the eye and hair color of the perpetrator. Now, a mass screening is taking place. The police have confidence in the phenotype predictions and can narrow down the pool of people being tested.
In another case, the police were dealing with UHR found in the sea. It was assumed that the body originated from one of the cargo ships belonging to a specific population. By using NGS for biogeographic origin estimation, we excluded that possibility, helping the police to focus on other theories.
And, of course, there are those cases that we are "bringing back to life" with FIGG. It is a work in progress. Our investigators have already expressed their excitement about FIGG. In some UHR cases, they felt there was no hope of moving forward.
Did you try conventional CE-based STRs first? What was the outcome?
Yes, we always go for the conventional CE-based STRs analysis first. Once we get the STRs, we do the CODIS search and, depending on the case, a familial search as well.
If we talk about the serial rapist case, we typed the autosomal and Y-STRs. Until recently we were getting the partial profiles only. We did finally get a mixture profile with the perpetrator being the main contributor, but no match in CODIS was found.
In case of the samples chosen for FIGG, we have the full STR profiles, but the database search didn’t result in any matches.
In your opinion, what difference will NGS make in these cases? Which challenges will it help resolve?
As the mass screening is taking place, it was very important for the police to get our confirmation for the eye and the hair color of the perpetrator.
When we talk about FIGG cases, NGS and FIGG are indeed bringing cases that have been on the shelf without any progress back to life, giving hope that they might be resolved.
What would you say to other laboratories considering implementing NGS for casework?
NGS really widens the range of the analysis and the data that can be extracted from a forensic sample. Not to mention, it is exciting to learn new techniques that can be implemented in the forensic DNA analysis.
