Microbes are ubiquitous and therefore can be found at all crime and death scenes

For forensic investigations, the predictable succession of microbes during decomposition can be leveraged to estimate the time since death, and microbial deposits in soil or from touch evidence can serve as trace evidence to link suspects and victims to crime scenes and personal objects. However, each of these forensic microbiome tools comes with a margin of error that reduces their reliability. Thus, an extensive knowledge GAP exists as the sources of these error margins are not fully understood. For microbiome-based versions of these forensic tools to be successful in criminal justice, microbial DNA collected during criminal investigations must accurately represent the microbial community present. When microbiome evidence is collected, the samples will include nonmicrobial (e.g., human) DNA, living microorganisms (e.g., bacteria) present in the environment, and relic DNA defined as extracellular microbial DNA leftover from dead cells along with DNA within cells that are no longer intact. Relic DNA can be preserved in the environment for extended periods of time, often in soils or other substrates, and can negatively impact microbial community diversity and species abundance estimations. These metrics are commonly utilized for forensic microbiome applications; therefore, the presence of contaminants, such as relic DNA, likely contribute to the error margins associated with microbiome tools. However, the extent to which relic DNA impacts microbial community analyses and the forensic microbiome is still not fully understood. Consequently, optimal and robust methodology for the generation of forensic microbiome data still needs to be refined to ensure reproducible and accurate results. The proposed research aims to improve data generation methods for forensic microbiome tools, such as PMI estimation and trace evidence analysis, through the inhibition of relic DNA, and will generate new knowledge on forensic microbiome processing that is applicable to all criminal and forensic investigations planning to incorporate reliable microbiome data into the criminal justice system. This project will provide fundamental knowledge and recommended best practices for the recovery of reliable and robust forensic microbiome data, building towards its accurate use in criminal cases. CA/NCF