The Future of Forensics

Human remains identification (HRID) is important in both crime scene investigation and in live human identification. Methods used to test degraded remains can also be applied to traditional cases involving DNA left by victims and perpetrators. Additionally, HRID is used to identify victims of mass disasters and military operations. To give an idea of the scale of this task, as of December 31, 2022, the US National Crime Information Center reported 8,242 active unidentified persons cases, as well as 546,568 open cases for missing people (1).

In forensic investigation, current pitfalls mostly lie with trying to type damaged or degraded samples. The most common practices for human identification revolve around short tandem repeat sequences, which require intact fragments of nuclear DNA. However, this is often not accessible for human remains, as time and environmental exposure break down DNA. Other methods are available; for example, exploring mitochondrial DNA, but this is not an easy piece of evidence to work with.  Many labs aren’t equipped to perform mtDNA testing and those that are cannot individualize with DNA of this kind.

I was lucky enough to have the opportunity to work with nanopore sequencing technology when the instrument was first released to a small group of researchers for testing. Given its unique characteristics, I saw its potential to revolutionize the industry. For example, the cost of the instrument is minimal compared with other sequencers, making it accessible to crime labs on a budget. Additionally, nanopore sequencing technology has the potential for in-field use thanks to its small size and weight, which would help in reducing backlogs and turnaround times. Some instruments also have limitless data generation, enabling simultaneous assessment of multiple targets. And that means we can make the most of our samples by performing various analyses, traditional short tandem repeat typing alongside single nucleotide polymorphisms, mtDNA, or epigenetic analysis, even when traditional methods yield unreliable results.

The gold standard in forensic analysis would be capillary electrophoresis (CE) and perhaps mtDNA sequencing, but I’m working to develop a more streamlined process that doesn’t rely on clunky traditional methods. With my team at the University of North Texas Health Science Center, I use nanopore sequencing technology for both whole genome and targeted analysis (post-PCR using standard commercially available forensic kits) (2).

We also designed RNA baits to target regions of interest – something that is new, but has resulted in increased enrichment of our target regions. Our results have shown improved discriminatory power from traditional methods because we are able to resolve isoalleles (alleles with the same length-based designation, but different sequence) that are indistinguishable through CE. One remaining challenge here is aligning allele names across different kits or platforms, which can lead to inconsistencies in our results compared with historical nomenclature standards. This remains a hurdle, as consistency in databases is essential in forensic work.

Working with a new technology has also given us plenty of bugs to work through, such as new basecallers and sequencing chemistry being released. Luckily, these aspects have stabilized over the past few years. We’ve also had to modify our library preparation methods to accommodate short DNA fragments that were clogging the nanopores. After undertaking several rounds of design and testing during development, we believe we now have the right baits to optimize results.

Moving forward, my team aims to expand upon our work with nanopore sequencing technology in forensics – working on a specific assessment of methylation for age estimation and body fluid identification. Nanopore sequencing technology platforms are able to conduct these tests without DNA modifications, which will help generate leads for HRID. However, for these systems to be a true competitor in the forensic community, they need a plug and play option for data analysis.

After working in this area for so many years, I still believe in building the capabilities of nanopore sequencing technology and its potential in forensics. I’d like to see a focus on resolving nomenclature issues in the future (which is currently being discussed), as well as consistent guidelines to be implemented across platforms. As this area progresses, I hope to see less of the same instruments in labs. Instead, with different specialized options available, labs will be able to select instruments that best fit their needs to ensure proper validations and cross-platform comparisons.