Advances in forensic technology continue to expand the availability and reliability of objective forensic information within the criminal justice system. The success of forensic testing and DNA database systems in aiding criminal investigations has often led to increased evidence submissions. With more submissions and the introduction of new technologies, we are continuing to see growth within the field. In the U.S. alone, the Bureau of Labor Statistics projects the number of jobs for forensic science technicians to rise by more than 11% by 2031.

With that in mind, this article discusses several of the many forensic tools and techniques that continue to change the way forensic laboratories work. Prominent examples include:

• Next Generation Sequencing (NGS), a massively parallel sequencing technology used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA;
• Forensic Investigative Genetic Genealogy (FIGG), which combines genealogical research with advanced DNA testing to provide potential links to unknown profiles;
• Activity level propositions, where analysts use Bayesian statistics to help address questions relating to transfer and persistence of DNA that might be of more relevance to the courts;
• Miniaturization, which includes shrinking laboratory equipment, and making the techniques faster (such as Rapid DNA technology), portable, and more efficient; and
• Artificial Intelligence, which has primarily been used in digital forensics, but is increasingly being used in other forensic applications including the designation of alleles and stutter within DNA profiles, and the assignment of the number of contributors to mixtures.

There are, of course, many more exciting areas of forensic DNA research, including lineage markers, proteomics, microbiomics, epigenetics, and non-human DNA applications. The emergence of these new technologies, coupled with continued support and development of probabilistic genotyping (PG) software for the interpretation of autosomal short tandem repeat (STR) profiles, has led our STRmix team to work with researchers and practitioners to enable the use of emerging technologies in casework. STRmix™ alone is now being used by over 100 organizations globally, with many more validating the software.

The emergence of so many new technologies onto the scene requires us to consider a range of issues relating to the current state of each technology. Given that, let me attempt to provide some food for thought.

Looking at Rapid DNA devices, STR profiles developed from such devices have been significantly more variable than standard laboratory methods to date. In 2018, the FBI established the Rapid DNA Taskforce which is working closely with developers of Rapid DNA technology to ensure profiles obtained from evidence collected at crime scenes are of suitable quality for CODIS entry. Inevitably, some of these profiles will be mixtures and appropriate PG solutions will be necessary. We have been working closely with end users and look forward to seeing the advances made with the updated kits and devices.

Existing PG software solutions assign likelihood ratios that address who might have contributed DNA to the sample (sub-source propositions). Quite often, however, the issue of interest to the court is not whose DNA is present, but rather how was that DNA transferred to an exhibit (for example, was it secondary transfer?) or how long might it have been there. These are activity level propositions.

Some analysts in Europe and Australia are already reporting activity level propositions in selected cases. Published research into the mechanisms of DNA transfer, persistence, prevalence, and recovery (collectively called DNA-TPPR) is increasing. These studies are particularly important since they can inform the probabilities necessary for the assignment of likelihood ratios at the activity level.

The wider use of PG software has led to the development and growing adoption of other tools designed to complete the full workflow from analysis to interpretation and database matching. Examples from the STRmix team alone include the data analysis software FaSTR™ DNA and DBLR™, a tool for the calculation of likelihood ratios for mixtures and single source profiles given any set of propositions including kinship scenarios.

FaSTR™ DNA is the STRmix team’s first foray into the use of artificial neural networks, which optionally can be used to assist trained analysts with the objective labelling of peaks within a profile probabilistically as artefactual or real. These probabilities subsequently can be used within STRmix™, meaning that not only alleles and stutters will be considered within the profile interpretation, but also artefacts including pull-up and baseline.

In addition to autosomal STRs, these tools now incorporate a growing NGS analysis capability, including within the investigative functions of DBLR™. DBLR™ can accept NGS sequence-based inputs and, when used in conjunction with STRmix™ software enables forensic laboratories to visualize the value of DNA mixture evidence, undertake mixture-to-mixture comparisons, conduct complex pedigree analyses, and achieve superfast database searches.

Looking to the future, the STRmix team anticipates more of the forensic community will evaluate the usefulness of NGS technology. We are actively engaging with the forensic community to gauge what this future looks like with regard to PG. Developers of this technology assert that it provides a depth of information beyond the capacity of traditional forensic DNA technologies. A key question, though, will be how it is used in combination with our legacy databases. Globally, existing databases used for criminal investigations contain tens of millions of STR profiles from known individuals. CODIS alone contains over 20 million offender and arrestee profiles. For this reason, we predict STR loci will be the workhorse of forensic laboratories for the near future.

One of the limitations of STR technology analyzed on capillary electrophoresis (CE) instrumentation is the relatively small number of loci available within the one multiplexed reaction. NGS technology does not suffer from this limitation. With STRs, additional loci can be squeezed into a multiplex horizontally by decreasing their length (using mini STRs, for example). Loci may also be added “vertically” by adding more dyes to the multiplex. This latter approach is a recent innovation from Promega with their Spectrum instrument and new PowerPlex™ 35GY System, encompassing both autosomal and Y-STR loci within the one multiplexed reaction using eight dyes.

Technology utilizing single nucleotide polymorphism loci (SNP) is now being embraced by laboratories routinely analyzing samples from small, degraded samples such as bone samples. Panels are being designed with tens or hundreds of thousands of SNP loci. These loci are much smaller than traditional STRs and are, therefore, less prone to degradation. Many of the advantages attributed to NGS technology relate to SNP loci.

By multiplexing thousands of SNP loci, increased discrimination power can be achieved, aiding investigation efforts such as facial reconstructions of human remains, the differentiation of identical twins, and the identification of previously unidentified remains using FIGG. Given the small sample sizes required, SNPs on NGS platforms are more sensitive and discriminatory compared with STRs analyzed on CE technology. They may produce additional information, such as identity-specific, ancestry, and phenotypic data.

The investment in NGS technology is significant, which may constrain the adoption of the technology for many laboratories in the immediate future. Reporting nomenclature for NGS-generated STRs will still need to be standardized, laboratory staff will still need comprehensive training, and proficiency testing programs will still be required. NGS technology produces significantly more data than traditional DNA typing using CE technology so it is likely laboratory information management systems will need significant configuration to account for this.

For laboratories that look to implement NGS technology for routine casework with a PG solution for interpretation of mixed DNA profiles, admissibility considerations will arise. There will inevitably be significant Daubert and Frye hearings and challenges to both profile generation and its interpretation. The forensic community will need to ensure that it is in a position to demonstrate that the evidence generated with NGS is based on scientifically valid reasoning which can properly be applied to the facts at issue.

The field of forensic biology is constantly evolving, and there are many new and exciting changes coming in the future. New techniques come with new challenges, and these challenges present exciting opportunities for research and collaboration in order to establish robust interpretation methods for emerging forensic DNA evidence. We look to the future with much interest, intrigued to see what else is around the corner.