Next-generation forensics is an emerging term used to coin the discipline’s adoption of next-generation methodologies, which include next-generation sequencing (NGS), next-generation mass spectrometry techniques, and instrumentation, setting new and highly sensitive limits of detection. Thanks to the adoption of these techniques and instrumentation, the field of forensic investigation is seeing steadfast growth and refinement.
The use of next-generation sequencing in forensics
In 2019, the National Institute of Justice (NIJ) reported on promising results on the use of NGS for human identification. Normally, genetic-based identification is conducted via PCR of short tandem repeats (STRs)—unique sequences found in each genome. But PCR amplification of STRs comes with challenges, such as copy errors that may confound interpretation. The traditional technique may also require multiple rounds of testing, which can be, as the NIJ points out, “cost- and time-prohibitive.” In contrast, NGS can sequence entire genomes with high accuracy. Furthermore, NGS has the added benefit of being powered by bioinformatics tools that markedly expedite STR analysis compared with standard PCR techniques.
The NIJ-funded research, led by Hanlee P. Ji from Stanford University, yielded a new genotyping methodology the Ji lab termed STR-Seq. STR-Seq is capable of analyzing nine separate samples for at least 439 STRs, each with 98 percent accuracy—based on R2 correlation analysis between the NGS reads and previously sequenced data. This is a remarkable feat considering that just genotyping with approximately 20 STRs is enough to warrant genetic identification in forensic science. This NGS-powered breakthrough drastically changes the boundaries of sensitivity to unequivocally and confidently derive the right conclusions.
Next-generation mass spectrometry approaches in forensics
Like PCR, forensics’ staple technology—mass spectrometry—continues to evolve. Whereas in the past, these instruments may have occupied a large space in the lab, the onset of the benchtop mass spectrometry analyzer in the 1980s drastically sped up analysis and eased laboratory work. In the 2000s, the development of LC-MS, which minimized the time for sample preparation without sacrificing accuracy, also significantly improved the sensitivities and throughput of toxicological analysis in forensics, as an example.
One recent encouraging breakthrough comes from the University of Surrey, where a team of researchers, led by M. J. Bailey, exploited matrix-assisted laser desorption ionization (MALDI) time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze the content of a single fingerprint from a global scale (mm) down to the pore (µm). By doing so, the team was able to identify the presence of cocaine metabolites at such sensitivities that they were able to tell apart the fingerprint of someone who had ingested cocaine from someone who merely grazed it. The results were published in May in the Analyst journal.
Such breakthroughs come in the nick of time. According to the NIJ’s 2021 Report to Congress, the heaviest burden forensics laboratories in the US continue to face is the ever-increasing workload in drug cases brought on by the opioid crisis. To put matters into perspective, the NIJ emphasized that “the result [of the overload in drug cases] is a substitution effect as resources are diverted from elsewhere in the laboratory to address casework in these areas.” With the emergence of new drug classes, the NIJ notes that the development of more sensitive technologies becomes more and more important. Technologies such as the aforementioned fingerprint analysis would highly increase throughput in the field.
However, despite these incredible advances, the NIJ points out some issues related to the preparation of analytes that may result in erroneous conclusions. The NIJ’s report highlighted that despite the fact that almost all (~90 percent) toxicological laboratories rely on mass spectrometry means to assess drug cases, more than 90 percent of these laboratories initially identify drug classes with immunoassays. However, the latter may fail to recognize newly emerging drugs such as fentanyl-related substances, synthetic cannabinoids, or synthetic cathinones (bath salts). As such, discovery of immunoreagents capable of identifying novel drugs may be in high demand.
“Like PCR, forensics’ staple technology—mass spectrometry—continues to evolve.”
Thankfully, this demand can be met through NGS and/or mass spectrometry-powered protein sequencing, both of which are routinely used in antibody discovery. Despite the fact that immunoassays are often resorted to for ease, mass spectrometry-based proteomics is not dependent on immunoassays. Thus, aside from discovering immunoreagents for forensics toxicologists, mass spectrometry-based proteomics and protein sequencing can be used to identify humoral immune response biomarkers or antibodies unique to each individual. The innate ability of antibodies to recognize microbial antigens makes them ideally suited as biomarkers of specific microbes, which would prove vital in cases of bioterrorism, for instance. Aside from the latter, proteomics may also be used to identify ethnic group, establish biological sex, and distinguish between individuals with a strand of hair, or even establish drowning as the cause of death by analyzing post-mortem blood biomarkers; and, by relying only on bone remains, proteomics may also estimate biological age, post-mortem interval, and discern between individuals. In 2019, protein sequencing was used to identify the ancient remains of a Denisovan man when DNA material was scant.
Proteomics and protein sequencing become especially relevant in DNA dead ends—when DNA information is scant or missing. Genetic material is also more fragile than proteins, and protein samples require less painstaking preparation for mass spectrometry analysis than nucleic acids do for PCR methods like NGS. Certainly, advances in mass spectrometry are set to pave the way for more accurate and sensitive collection of data in forensics investigation.
Such is the case for another forensics field, forensics entomology, which has witnessed recent breakthroughs in mass spectrometry analysis of insects. Traditionally, identification of insect species is performed to determine the post mortem interval or estimated time of death. Post mortem infestation of the body has been associated with specific species of insects in different environments, and at different timepoints. This knowledge has been exploited since the thirteenth century in China, where the first forensics entomology case was recorded. However, conventional insect identification is cumbersome, not just due to laborious constraints, but also because viable sample preservation is vital. Since sample collection may involve preserving insects in formaldehyde or ethanol, it is not always feasible to grow larvae to maturity for species determination, nor helpful for PCR analysis as DNA may acquire lesions over time.
To address this problem, the Musah lab from the Department of Chemistry at the University at Albany proposed a method to analyze the proteomics profiles from several species from a single sample using direct analysis in real time-high resolution mass spectrometry (DART-HRMS). In their study, the researchers analyzed ethanol-preserved maggots through a DART ion source attached to a ToF analyzer in positive ion mode. Using machine learning, they processed the spectral data and developed a prediction model for multi-species samples with prediction confidence levels ranging between 80 to 99 percent. The Musah lab’s proof-of-concept findings, published in February 2020 in Analytical Chemistry, illustrate the potential of mass spectrometry in automating and facilitating otherwise painstaking tasks in forensics with precision.
The evolution of forensic investigation
Perhaps the three most notable technological advances that have disrupted forensic investigation are: 1) the introduction of DNA sequencing, 2) the development of immunoassays, and 3) the ushering in of benchtop mass spectrometry. Today, forensics encompasses several disciplines, including forensic science, forensic toxicology, forensic entomology, and forensic pathology, to name a few. In all of the latter disciplines, the use of mass spectrometry has and continues to play a defining role. With the advent of more comprehensive and sensitive approaches, mass spectrometry is set to usher in a new era of next-generation forensics.