Developmental Validation of a Novel Multi-analyte Recovery Method for Trace Biological Samples

The forensic scientist’s toolbox will soon include an entire suite of molecular analytes and there is an urgent unmet need for a multianalyte recovery method compatible with trace biological samples. While the target for STR genotyping and human individualization is clearly DNA, there is a growing need for analysis of multiple analytes in forensic casework. For example, analysis of mRNA is proving to be one of the best methods for identification of body fluids which is critically important in sexual assault casework and in associating a body fluid to a DNA profile. Analysis of proteins is also useful for body fluid identification and for individualization in highly compromised samples with little recoverable DNA using Genetically Variant Peptides (GVPs). Analysis of targeted metabolites is routinely performed in forensic toxicology laboratories but analysis of all metabolites in a sample (metabolomics) has potential in determining cause of death and the Post-Mortem-Interval. On a prior NIJ award, the proposal team has developed a novel method which enables highly efficient recovery of DNA (including degraded DNA) and RNA from trace biological samples but is also non-destructive to proteins and metabolites. In this project, the same team proposes to perform a full developmental validation of this method according to SWGDAM guidelines. Using Capillary Electrophoresis-based STR DNA profiling, Next Generation Sequencing-based targeted RNA sequencing, and Mass Spectrometry-based targeted protein and spiked metabolite analysis, recovery of analytes will be assessed in terms of efficiency and compatibility with downstream assays. Parameters such as sensitivity, stability, reliability, and reproducibility will be evaluated using body fluids of relevance to sexual assault casework and using surface swabs from challenging surfaces such as brass firearm cartridges. Streamlined protocols for multianalyte recovery from body fluid stains and surface swabs typically encountered in forensic casework will also be established, including a novel swabbing method for recovering trace DNA from challenging substrates. Large numbers of case type samples will be used for recovery of DNA alongside validated commercial kits to demonstrate non-inferiority of this method, which is uniquely also non-destructive to other analytes, allowing analysis of all the molecular information within a sample. The proposal team will work with forensics practitioners to ensure new protocols align with existing workflows. At the conclusion of the program, deliverables include detailed reports on performance and dissemination of protocols that will be shared via scientific publication and presentations. CA/NCF